新規試験法提案書

腐食性試験代替法

平成 20 年 8 月

国立医薬品食品衛生研究所

ヒト皮膚モデルを用いた皮膚腐食性試験代替法の評価報告書

JaCVAM 評価会議

平成 20 年（2008 年）6 月 30 日
平成 23 年（2011 年）4 月 20 日改定

1JaCVAM 評価会議
井上 達（国立医薬品食品衛生研究所 安全性生物試験研究センター）
林真（財団法人食品農医薬品安全性評価センター）

田中憲穂（食品薬品安全センター 秦野研究所）
吉田武美（昭和大学薬学部）
溝口昌子（聖マリアンナ医科大学）
佐神文郎（エーザイ株式会社／日本製薬工業協会）
岡本裕子（株式会社コーセー／日本化粧品工業連合会）
小野寺博志（医薬品医療機器総合機構）
五十嵐良明（国立医薬品食品衛生研究所 環境衛生化学部）
任期：平成 19 年 1 月 1 日～平成 20 年 12 月 31 日
西川秋佳（国立医薬品食品衛生研究所 安全性生物試験研究センター）
田中憲穂（食品薬品安全センター 秦野研究所）
吉田武美（昭和大学薬学部）
横関博雄（東京医科歯科大学）
吉村 功（東京理科大学）
渡部一人（日本製薬工業協会）
岡本裕子（日本化粧品工業連合会）
大島健幸（日本化学工業協会）
小野寺博志（医薬品医療機器総合機構）
小笠原弘道（医薬品医療機器総合機構）
吉田 緑 （国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部）
五十嵐良明（国立医薬品食品衛生研究所 生活衛生化学部）
長谷川隆一（独立行政法人 製品評価技術基盤機構）
浅野哲秀（元日東電工株式会社）
任期：平成 22 年 4 月 1 日～平成 24 年 3 月 31 日
オブザーバー：JaCVAM 運営委員
大野泰雄（国立医薬品食品衛生研究所）
中澤憲一（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
増田光輝（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
小島 肇（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
板垣 宏（日本動物実験代替法学会会長）
任期：平成 19 年 1 月 1 日～平成 20 年 12 月 31 日
大野泰雄（国立医薬品食品衛生研究所）
関野祐子（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
増田光輝（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
小島 肇（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）
秋田正治（日本動物実験代替法学会）
柴辻正喜（厚生労働省 医薬食品局 審査管理課 化学物質安全対策室）
2

実国慎一（経済産業省 製造産業局 化学物質安全対策室）
任期：平成 22 年 4 月 1 日～平成 23 年 4 月 30 日
以上3

3 次元培養ヒト皮膚モデル（以下、皮膚モデルと記す、Vitrolife-SkinTM）を用いた腐食性試験の動物実験代替法について、第三者評価委員会からの報告を受け１）、以下の 8 項目について審議した。
本項目は OECD ガイダンス文書 No．34 に示された検討項目である２）。なお、本動物実験代替法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。
＜審議内容＞
１．検討対象の試験法とその妥当性を示すデータは、透明で独立な評価を受けているか。
・当該試験法は論文に発表されており、透明性は高い。
・バリデーションデータは、透明性の高い独立した第三者評価を受けている。
２．当該試験法で得られるデータは、対象毒性を十分に評価あるいは予測できるものであるか。デー
タは、当該試験法と従来の試験法の、代替法としての繋がりを示しているか。あるいは（同時に）そのデータは、当該試験法と、対象としているあるいはモデルとしている動物種についての影響との繋がりを示しているか。
・ in vivo と in vitro の一致率は 83．3％であった。乳酸や 5％水酸化カリウム水溶液で不適合があった。
・一致しなかった物質については、必ずしも in vivo の表示が適切でないとの指摘があり、調査が必要であるとされた。
・ヒトの腐食性を予測できていると考えられる。
OECD のガイドライン 431 に掲載されている既存の試験法 EpiDermTM と比べて３）、基本的に同じ作用機構を持ち、かつ同等の性能を持つという評価がなされた。
腐食性を細胞毒性で予測することの妥当性について、腐食性物質が接触すると角質層に吸収、拡散
して通過後、下層の細胞に障害を及ぼし、障害の程度は表皮細胞の生死を指標とするので、この方法
は腐食性を予測する条件を満たしているとの見解が示された。
３．当該試験法は、ハザードあるいはリスク、あるいはその両方を評価するのに有用であるか。
ハザードの同定には有効である。
４．当該試験法とその妥当性を示すデータは、その試験法で安全性を保証しようとする、行政上のプログラムあるいは関係官庁が対象としている化学物質や製品を、十分広く対象としたものとなっているか。当該試験法が適用できる条件及び適用できない条件が明確であるか。
・ 今回のバリデーションでは、毒劇法の対象となる被験物質と判定基準物質が使われている。
・ 腐食性の作用機構を判断するに足る必要最小限のデータが得られている。
・ 被験物質が強アルカリ性の場合、培養細胞の基盤であるコラーゲンマトリックスを溶解する。着色性物質や吸着性が強い物質は適用が困難である。
５．当該試験法は、プロトコルの微細な変更に対して十分頑健で、適切な訓練経験を持つ担当者と適切な設備のある施設において、技術習得が容易なものであるか。
習得について大きな問題はない。実施においても特別な技術は必要ない。なお、測定キットであ4ることからプロトコルに大きな変更の余地はない。
６．当該試験法は、時間的経費的に有用性があり、行政上で用いられやすいものであるか。
効果的である。
７．当該試験法は、従来の試験法と比べて、科学的・倫理的・経済的に、新しい試験法あるいは改訂
試験法であることが正当化されているか。
正当化されている。
８.安全性評価のための行政的資料として、 受け入れ可能な試験法であるか。
化学物質による直接的な腐食性を評価できる方法である。その範囲において、行政的な利用は可能である。
化粧品・医薬部外品の腐食性の評価ならびに、毒物及び劇物取締法における毒物又は劇物の区分のための試験法として、利用可能である。
以上の審議の結果、JaCVAM 評価会議は、動物実験代替法である皮膚モデル（Vitrolife-SkinTM）を用いた腐食性試験を定められた方法で適切に利用すれば、化学物質の腐食性を科学的に評価できると
結論した。
参考文献
１．皮膚腐食性試験代替法のための第三者評価委員会報告書
２．OECD (2005) OECD Series on testing and assessment Number 34, Guidance document on thevalidation and international acceptance of new or updated test methods for hazard
assessment, ENJ/JM/MONO(2005) 14
３．OECD(2004) OECD guideline for the testing of chemicals, Test guideline: 431, in vitro Skin
Corrosion: Human skin model test5

ヒト皮膚モデルを用いた皮膚腐食性試験代替法の第三者評価報告書

評価対象品目： Vitrolife-Skin™（グンゼ株式会社）

皮膚腐食性試験代替法の第三者評価委員会
評価委員長
委員
委員

小野寺博志（医薬品医療機器総合機構）
岡本裕子（株式会社コーセー／日本化粧品工業連合会）
五十嵐良明（国立医薬品食品衛生研究所 環境衛生化学部）

平成 20 年 6 月 10 日

1

略号
ECVAM：European Center for the Validation of Alternative Methods
GLP: Good Laboratory Procedure
ICCVAM： Interagency Coordinating Committee on the Validation of
ICH：International Conference on Harmonization
OECD：Organization for Economic Co-operation and Development
SOP: Standard Operation Procedure

2

Alternative Methods

要旨
ウサギを用いる皮膚腐食性試験の代替として提案された 3 次元培養ヒト皮膚モデル（以下、皮膚モデルと記す）Vitrolife-Skin™を用いる試験法の有用性を評価した。
3 次元培養ヒト表皮モデル EpiDerm™
を用いた皮膚腐食性試験法は既に欧米でバリデーションが終了し、OECD ガイドラインになっている。
Vitrolife-Skin™は我が国で開発された EpiDerm™と類似の皮膚モデルであり、線維芽細胞を含むコラー
ゲンからなる真皮層の上に角質層を持つ表皮層が重層化された構造を有している。今回 EpiDerm™とVitrolife-Skin™を同時に被験物質の数を最小限にして検討する Catch up validation によって評価した。Vitrolife-Skin™を用いた試験及び結果の判定法は EpiDerm™とほぼ同様で、被験物質で処理した後、染色し、吸光度を計測することによって細胞生存率を求め、腐食性の程度を判定する。試験に用いた被験物質（12 種）は既に in vivo で腐食性の評価がされている物質で、試験実施施設（6 施設）には名称を隠しコード化して供与された。試験は事前に講習を受けた実施者が SOP に従い実施された。試験は 2 回繰り返し、判定結果が異なる場合は 3 回目の試験を実施し、多数決での判定を結果とした。
測定値はいずれの物質も近似値を得ることができ、施設内の再現性は高いものと判断された。EpiDerm™
においては 12 物質中 1 物質で 1 施設が異なる判定結果を示したが、Vitrolife-Skin™では全ての施設で判定結果は同じであった。また、判定に苦慮した物質は EpiDerm™モデルと Vitrolife-Skin™モデル
で同一物質であり、施設間での再現性は極めて高かった。以上の結果より、Vitrolife-Skin™を用いた
皮膚腐食性の評価は、特異性、感度、再現性の点において EpiDerm™と同等あるいはそれ以上の識別能力を有すると判断した。一方、モデルの支持組織であるコラーゲンに影響を及ぼす強アルカリ性物質
等は判定に支障があること、今後、大量生産の際の製造方法の変更、それに伴う品質管理や同等性等確認の方法が課題として挙げられた。しかしながら、Vitrolife-Skin™は EpiDerm™同様、動物を用いな
いこと、短時間で結果が得られること、コスト面では EpiDerm™と比べ安価であること、また日本で製造販売されるため安定した供給が可能である利点が挙げられた。以上、VitroLife Skin™を用いる皮膚腐食性試験は動物実験代替法として有用であると評価した。

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評価結果
1. 試験法の科学的及び規制面からの妥当性
皮膚腐食性試験は皮膚刺激性試験の一環として行われ、種々のガイドラインでは Draize らにより提唱されたウサギを用いる方法が推奨実施されている。この方法は被験物質の皮膚刺激性を検出する感度は非常に優れているものの、判定を肉眼で行うため客観性に乏しく実験間や施設間での再現性が乏しい、更に動物に激しい苦痛とストレスを与えることが社会的に問題となり、以前より動物を使用しない動物実験代替法（以後、代替法と記す）の開発が切望されていた。OECD ガイドライン 431 には、皮膚腐食性試験として角質層を有し再構築された表皮から構成された皮膚モデルの記載がなされている。この試験法は、腐食性物質が角質層に吸収された後拡散し、下層の細胞に障害を及ぼすという現象をもとに、被験物質接種後の細胞生存率を指標に皮膚腐食性を評価している。これら EPISKIN™や
EpiDerm™等の 3 次元培養ヒト表皮モデルは欧米で既にバリデーション試験が実施され、欧州では化学物質の皮膚腐食性評価を目的として承認され、化学物質のリスク表示識別等に採用されている。
我が国で既存の化学物質を評価する場合、OECD で承認された試験方法による結果を受諾することは可能であるが、現在まで代替法での結果をもとに評価された例は多くない。安全性評価における代替法の普及が切望されている現状において、我が国でも積極的に受け入れることが必要となっている。
また一方で、国内企業からも国際的な評価に耐えうる代替法が開発されることも推測される。その場
合、バリデーション等が実施され、十分な有用性と再現性が証明されることにより代替法としての受け入れも可能となる。
今回、我が国で安定な供給が期待できる皮膚モデルが開発され、これを用いた皮膚腐食性試験代替法の有用性についての検討が必要となった。
Vitrolife-Skin™はグンゼ(株)によって開発された線維芽細胞を有するコラーゲンからなる真皮層の上に角質層を持つ表皮層が重層化されている 3 次元培養ヒト皮膚モデル（以下、皮膚モデルと記す）である。類似の製品として EpiDerm™があり、これはポリカーボネート膜上に角質層を持つ表皮が構築されたモデルである。これらの皮膚モデルは基本的に角質表皮層を構築させる基盤が異なるのみで、被験物質は角質層を通過し表皮細胞に作用して細胞障害を起こす。さらに、いずれの皮膚モデルにおいても細胞生存率の測定に用いる色素は同じ 3-（4,5-dimethylthiazol-2-yl）-2,5 tetrazolium
bromide (MTT)で、皮膚腐食性の評価機構において本質的な違いはないと考えられる。
OECD（2005）ガイドライン 34 の Catch-up バリデーション研究の定義では、既に妥当性の確認された方法と同じ原理による方法であれば、新規の方法であっても信頼性の高い施設で適切な方法でバリ
デーションを実施すれば、多数の被験物質と多施設による大規模なバリデーション実施することなく、
小規模のバリデーションで評価可能とされている。今回実施されたバリデーションでは、細胞構成や機能が同等と考えられる Vitrolife-Skin™と EpiDerm™について、同じ被験物質を同時期に試験していることから Catch-up バリデーション研究に該当すると考える。これら２つのモデルを用いた皮膚腐食性試験結果で同等性を確認できるならば、Vitrolife-Skin™が EpiDerm™と同等の有用性を持つことが証明される。
また今回の実施報告は、EpiDerm™用いた皮膚腐食性試験代替法の我が国における評価としての意味をもつと考える。
2. 試験プロトコール構成の妥当性
実 施 さ れ た 試 験 プ ロ ト コ ー ル は EpiDerm™ を 用 い た バ リ デ ー シ ョ ン 研 究 と ほ ぼ 同 じ で あ る 。

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Vitrolife-Skin™では前培養法及び染色液の容量、組織からの抽出法に違いがあるのみで、基本的な曝露時間と判定基準については同一のものを用いている。被験物質は角質層を通過して表皮細胞に作用し、MTT の取り込み量より求めた細胞生存率の減少から皮膚腐食性を判定する。
プロトコールの概略は以下のとおりである。6 wellプレートの各wellに培養液 1 mLを加えて皮膚モ
デルを置き、被験物質が液状の場合はピペッターで 100 µL、粉末など固形の場合は 100 mgを計測して上層に適用した (n=2)。固形物質の場合は適用後浮遊を防ぐ目的で画鋲等の頭で軽く押さえた。被験物質を 3 分または 60 分処理後、プレートから被験物質をデカンテーションにより除去した後、10 mLのPBSで 2〜3 回軽く洗浄した。洗浄後、ペーパータオル等で水分を切り、破損に注意しながら別の 24
wellプレートに皮膚モデルを移動した。EpiDerm™モデルの場合MTT色素を含む培養液を皮膚モデルの下方に 0.3 mL、Vitrolife-Skin™モデルの場合は上方から 1 mL加えた。37℃、CO2インキュベータ中に 2〜3 時間静置した後、Vitrolife-Skin™は直径 6 mmの円形状にくり貫き、以下の抽出を行った。イソプ
ロパノールをEpiDerm™モデルでは 2 mL、Vitrolife-Skin™では 1 mL加えた。一晩冷蔵庫で放置、96 well
プレートに抽出液を 200 µLずつ移し（1 物質あたり 1〜3 well）、マイクロプレートリーダーを用いて
540 nmあるいは 570 nm領域での吸光度を測定した。イソプロパノールのみを加えたwellをブランクとし、実測値とブランク値の差を求めた。溶媒対照の吸光度を 100％とし各物質の 3 または 60 分間処理
時の％を計算した。3 分間処理したときの生存率が 50％未満、あるいは 3 分間では生存率が 50％以上であるが 60 分間処理したときに 15％未満の結果を示す物質を腐食性ありと判定した。一方、3 間処理したときに生存率が 50％以上、60 分間処理したときに 15％以上の物質は非腐食性と判定した。試験は2 回繰り返し、2 回の試験で判定が異なった場合は追加の試験を実施し、優位の判定結果をその物質の最終評価とした。
3. バリデーションに用いた物質の分類、選択理由の妥当性
今回実施した Virolife-Skin™モデルについて、被験物質の数 12 は ECVAM などで実施された EPISKIN™
モデルや EpiDerm™モデルのバリデーションと比べ少ないが、Catch-up バリデーションを採用することで評価は可能と考えた。
予備試験では 10％水酸化ナトリウム溶液(potassium hydroxide(10%))及び塩化ベンザルコニウムを陽性対照物質として選定して手技の確認がなされたが、本試験での陽性対照物質は 10％水酸化カリウ
ム水溶液を用いた。本試験では陰性対照物質として 20％ラウリル硫酸ナトリウム溶液［sodium laurylsulfate(20%)］を用いた。被験物質の選定に当たっては ECVAM でのバリデーションで用いられた物質を基本とし、本邦の法規制（毒劇法）を考慮しつつ、物性的なバランスも考慮した。12 種類の性状は液体 6 種、固体 4 種、粉末 2 種であった。

In vivo での皮膚腐食性の評価は Botham ら (1995) のデータをもとに分類した。腐食性物質は硫酸(10%) [sulfuric acid(10%)]、octanoic (caprylic) acid、水酸化ナトリウム溶液(4.88%)[sodiumhydroxide (4.88%)]、フェノール(phenol)、酸化クロム(Ⅵ) (無水クロム酸、chromium trioxide、
chromium(Ⅵ) oxide が正式名）及びりん酸 (phosphoric acid）の 6 種、非腐食性物質として 6 種、過ほう酸ナトリウム四水和物（sodium perborate、sodium peroxoborate tetrahydrate が正式名）、テト
ラクロロエチレン(tetrachloroethylene)、水酸化ナトリウム溶液(5%) [potassium hydroxide(5%)]、4-amino-1,2,4-triazole 、 L- 乳 酸 (L-lactic acid) 及 び イ ソ プ ロ パ ノ ー ル (isopropanol,

別名2-propanol) の 6 種である。
次に被験物質を化学構造で分類すると、無機酸の腐食性物質は sulfuric acid(10%)、非腐食性物質

5

は phosphoric acid、無機塩基の腐食性物質は sodium hydroxide (4.88%)、非腐食性物質として
potassium hydroxide (5%)と sodium perborate、有機酸の腐食性物質として octanoic acid、非腐食性 物 質 は L-lactic acid 、 有 機 塩 基 の 腐 食 性 物 質 は フ ェ ノ ー ル 、 非 腐 食 性 物 質 は4-amino-1,2,4-triazole、金属塩の腐食性物質は chromium trioxide、中性の非腐食性物質溶剤として
tetrachloroethylene 及び isopropanol と、選択された物質は各種物性がほぼ均等に選択されている。
表 1．被験物質
番号

被験物質名

腐食性*

コメント

規制
劇物

物

性

1

Potassium hydroxide (10%)

C

陽性対照物質液体

2

Sodium lauryl sulfate (20% )NC

陰性対照物質液体

3

Sulfuric acid (10%)

C

劇物判定薬

非劇物 液体4Octanoic (Caprylic) acidC

5

Sodium hydroxide(4.88%)

C

劇物判定薬

非劇物 液体6

Phenol

C

劇物判定薬

劇物

固体 mp 40-43℃

7

Chromiun trioxideC

劇物

固体 mp 197℃

8

Phosphoric acidC

非劇物 固体 mp 42.3℃

9Sodium perborateNC

非劇物 粉末 60℃で分解

10 TetrachloroethyleneNC

非劇物 液体 bp 121℃

11 Potassium hydroxide (5%)

NC

液体

12 4-Amino-1,2,4-triazoleNC

非劇物 粉末 mp159℃

13 L-Lactic acidNC

非劇物 固体 mp 52.8℃

14 Isopropanol (2-propanol)NC

非劇物 液体 bp 82.5℃

非劇物 液体 bp 239.7℃＊ C: corrosive, NC: non-corrosive〔Botham, et al., 1995〕
4. 試験法の正確性を評価するために用いられた物質の in vitro 及び参照データの有無選定した 12 種類の被験物質の多くは ECVAM、ICCVAM で EpiDerm™、EPISKIN™あるいは Corrositex™を用いた皮膚腐食性試験バリデーションで使用された物質である。これら被験物質の評価結果は下記の資料として提出されている。
Liebsch et al., ATLA 2000; Barratt et al., Toxicol. in Vitro 1998; Fentem et al, Toxicol.
in Vitro 1998; Worth et al., ATLA 1998; Botham et al., ATLA 1995
ICCVAM (2002) NIH Publication No: 02-4502
ICCVAM(1999) NIH Publication No:99-4495
本邦における「毒物及び劇物取締法」（毒劇物法）で劇物指定の際の判定基準となっている物質が陽性対照を含め 3 物質あり、いずれも腐食性物質である。しかし、劇物指定外物質でも腐食性を認める物質がある。毒劇物法では 10％濃度で腐食性を示すものを規制対象としており、試験研究からの invivo 評価とは異なる。これは皮膚腐食性試験が物質としてのポテンシャルを評価する方法であり毒劇物指定条件の違いがある。今回は毒劇物の指定条件に限定せず、純粋に腐食性の評価を行った。

6

5. すべてのデータ及び結果
各施設で実施された本試験 2 回及び追試験の全ての結果(62 回)を、実験施設、実験者、試験種類（回数）、記録日、使用モデル名、測定波長とともに生の吸光度をデータシートに記入して提出されており、これらを用いて評価した。EpiDerm™モデルにおいては腐食性物質を正しく腐食性と判定した例は、陽性対照物質を除く６被験物質の 30 試験中 29 試験で、感度(sensitivity)は 29／30(96.7％)であった。
一方、非腐食性物質を正しく非腐食性と判定した例は６被験物質 30 試験中 20 試験で、特異性(specificity)は 20／30（66.7％）であった。非腐食性を腐食性と誤認判定した物質は potassiumhydroxide (5%)と L-lactic acid で、全ての機関で判定が異なっていた。陽性予知能力(positive
predictivity)は 39 の陽性事例のうち 29 例（74.4％）、陰性予知能力は 21 例中 20（95.2％）であった。
合計すると正しい結果が得られた割合（一致率）は 60 試験中 49 例（81.7％）であった。腐食性物質
である sulfuric acid を 2 回とも陰性と判定した施設が 1 施設あった。
Vitrolife-Skin™モデルでの感度は 30／30（100％）、特異性は 20／30（66.7％）、陽性予知能力 30／40（75％）、陰性予知能力 20／20（100％）であり、EpiDerm™モデルと同等の判定率であった。非腐食性を誤って腐食性と判定したのは potassium hydroxide (5%)と lactic acid と EpiDerm™モデルと同じ物質であった。
表 2 EpiDermTMの一致数
Vitro

Corrosive

Non-Corrosive

29

1

Vivo
Corrosive

10 (5% KOH,

Non-Corrosive

20

Lactic Acid)

表 3 Vitrolife-SkinTMの一致数
Vitro

Corrosive

Non-Corrosive

30

0

Vivo
Corrosive
Non-Corrosive

10 (5% KOH,
Lactic Acid)

20

次に、12 種の被験物質について皮膚モデルでの判定結果と in vivo のデータを比較した。明らかな腐食性物質（sulfuric acid）についてある 1 施設が異なった判定をしたため、sulfuric acid については 5 施設での結果を最終判定とした。被験物質数を分母として起算すると、EpiDerm™モデル及びVitrolife-Skin™モデルの感度は、両モデル共に 12/12(100％)、特異性 4/6(66.7％)、正確性 10/12（83.3％）、偽陽性率 2/12(16.7％)、偽陰性率 0/12(0％)といずれの結果も同一であった。よって、
EpiDerm™及び Vitrolife-Skin™両モデルの間に腐食性の評価に差はないと考えられる。ECVAM で実施された EpiDerm™を用いた皮膚腐食性試験のバリデーション結果と比較してもほぼ同等で、特に、偽陰性率が少ない点で EPISKIN™よりも予測性は高いと評価した。

7

表 4. EpiDermTM及びVitrolife-Skin TMの特異性、感度、一致性の比較（国立衛研、sulfonic acid を除外して計算）
EpiDermTM

Vitrolife-SkinTM

EpiDerm(ECVAM)EPISKIN(ECVAM)

12

12

24

60

感度

100％（12/12）

100％（12/12）

92%

82％

特異性

66.7％（4/6）

66.7％（4/6）

83%

84％

正確性

83.3％（10/12）

83.3％（10/12）

92%

83％

偽陽性率

16.7％（2/12）

16.7％（2/12）

17%

16％

偽陰性率

0％（0/12）0％（0/12）

8%

18％

試験物質数

本試験において、L-lactic acid は、非腐食性物質(NC)として分類し皮膚モデルとの判定結果を比較したが、EpiDerm™及び Vitrolife-Skin™を用いた試験では腐食性物質(C)と判定し偽陽性物質として区分された。Liebsch et al.(2000)の報告では L-lactic acid の in vivo classification は R34、すな
わち腐食性ありとしている（彼らは一方で EpiDerm™モデルでは NC としているが）。Barratt et al.
(1998)の ECVAM Workshop での報告にも同様の結果がある。
したがって、今回の L-lactic acid の in vivoの判定の根拠については確認をとる必要があり、もし in vivo で腐食性が示されれば、皮膚モデルでの陽性の結果は正しいことになる。
Potassium hydroxide は 10%溶液を陽性対照として用い、5％溶液を in vivo 陰性物質として評価している。In vitro ではいずれの皮膚モデルも 5％溶液は腐食性ありと判定し、偽陽性物質との分類となる。しかし、NIH Publication No:99-4495,（1999）では in vivo での判定は discordant（一致していない）との記載がある。試験濃度により判定が異なることは予測範囲内であり、本結果は皮膚モデ
ルでの検出感度が高いことによる可能性を示唆している。
6. 試験法の正確性（再現性、頑健性）
試験結果に影響を及ぼす要因としては被験物質の適用時間と洗浄方法であり、一連の操作で高度の技術を要求されることはない。吸光度測定はマイクロプレートリーダーで行い、細胞生存率の算出は
溶媒対照を基準値とし、その比率として表される。
同一施設内で 2 回試験を繰り返し、1 回目と 2 回目で異なる結果を得たのは、EpiDerm™では 12 物質
中 sulfuric acid と L-lactic acid の 2 物質であり、それぞれの物質につき 2 施設で観察された（施設１の実験番号 48、施設 2 の 14、施設 3 の 15、施設 5 の 51）。Vitrolife-Skin™の場合、sulfuric acid,octanoic acid, sodium hydroxide の 3 物質で観察された（施設 2 の実験番号 34、施設 3 の 15 及び 25）。
同一物質で 2 回の試験で異なる判定結果が生じる割合は 60 の試験回数のうち EpiDerm™で４回(6.67％)、
Vitrolife-Skin™で 3 回(5.0％)と、Vitrolife-Skin™モデルの方がわずかに低かった。判定が異なる理由としては細胞生存率が判定基準値近傍の値を示したためであるが、基準値の区切りにより判定が異なる場合においても細胞生存率は 1 回目、2 回目、追加試験ともほぼ同じ値を示し、同一施設内での再現性は高かった。
EpiDerm™を用いた場合、施設１のみ sulfuric acid の最終判定が他施設とは異なっていた。施設間での判定一致率は 1/6(16.7％)（バリデーション報告書では 1/5 とあるが、6 施設あるので誤記と思われる）、物質間での判定不一致率では 12 物質中 1 物質となる。Vitrolife-Skin™を用いた場合、各施設間での被験物質に対する最終判定結果は全て一致した。
以上のように、施設内及び施設間での再現性に関しては、Vitrolife-Skin™を用いた方が EpiDerm™
8

に比べ良好の成績が得られている。
被験物質数を分母として起算した EpiDerm™及び Vitrolife-Skin™での感度は 12/12(100％)、特異性
4/6(66.7％)、正確性 10/12（83.3％）、偽陽性率 2/12(16.7％)、偽陰性率 0/12(0％)でいずれの項目でも両モデルの数値は一致し、差異を認めなかった。24 種の物質を対象にして ECVAM で実施された
EpiDerm™のバリデーション研究の結果と比較した場合、Vitrolife-Skin™は特異性と正確性においてやや数値が低いものの、偽陰性率は 0 であった。60 種類の物質について行われた EPISKIN™の結果と比べると特異性の面では劣るものの、他の評価項目では優れていた。よって、EpiDerm™は EPISKIN™と比べて腐食性の予測モデルとして優れていると考えた。試験に用いた物質数や種類が異なっているため一元的な優劣の評価は困難であるが、今回の結果だけで評価すると Vitrolife-Skin™は現時点で最も予測性の高いモデルとして確立されている EpiDerm™と同等の性能を有すると思われた。
7. 試験法の信頼性
試験法は EpiDerm™を用いたバリデーション研究で実施されたものと同一のプロトコールを用いている。 試験実施前に、標準操作手順書(SOP)を作成し試験実施者に対する技術講習会を開催し技術移転を行っている。
用いた皮膚モデルは各メーカーから直接実験施設に供与された。供与数は予備試験 1 回、本試験 2回とし、それぞれ 2 キットを使用した。追加試験の実施が必要なときは、別途追加のキットが供与された。皮膚モデルは試験開始まで保管、または添付された培養液で前処理された。Vitrolife-Skin™については前培養期間が 0 あるいは 1〜2 日前と幅があり、この前処理時間に関する影響について明らかではない。
バリデーション実行委員会は被験物質の皮膚腐食性を調査して選定し、各施設にコード化して配布した。そのため、実際の実施者は試験物質がブラインド化され判別不能となっている。被験物質の物
性（液体・固体）により適用法を変え、固形の場合、画鋲の後ろで押さえる方法がとられている。しかし、画鋲に限定せず、規格や性状の影響も考慮し、押さえるものの形や大きさ等を記載した方がよかった。
Vitrolife-Skin™は被験物質が強アルカリ性の場合、基盤となるコラーゲンスポンジが溶解し、適用部位をパンチでくり抜くことが不可能なため、色素の抽出の際に支障が生じた。今回タンパク変性を有する物質は腐食性と判定しているが、今後こうした場合の判定基準についても SOP に記載すべきである。
細胞生存率(％)はマイクロプレートリーダーで吸光度を測定し、溶媒対照の吸光度と直接比較する
ことによって求められる。判定基準値はあらかじめ設定されており、腐食性の評価に対して主観性の入るところはない。
試験は同じ被験物質で 2 回繰り返して行い、それぞれの試験判定が異なった場合は追加試験を実施
し、最終試験結果を被験物質の評価とした。2 回の試験で異なった結果が生じる割合は EpiDerm™で 4／60(6.67％)、Vitrolife-Skin™で 3／60(5.0％)でほぼ同程度であった。基準値の境界付近で判定が分かれた場合でも、生存率は 1 回目と 2 回目、追試験ともほぼ同じ値を示した。
8. データの質試験実施施設すべてが GLP に準拠しているわけではない。しかし、試験実施者は、技術講習会に参加し技術移転を受けた者に限定し、SOP に沿って実施された。皮膚モデルは製造会社から品質が保証さ9れているものと同一キットが直接実施者に配送され、被験物質は一機関からコード化されたものが送付された。コードの開示は全施設から結果収集が終了したのち行われ、盲検性について信頼性の向上に努めた。
試験結果のまとめは所定の記録用紙（入力欄）が作成され、実施施設名、記録者、測定波長、被験物質番号と吸光度を皮膚モデル毎に入力する様式がとられた。吸光度データシート（結果確認欄）は吸光度から計算された各被験物質の生存率とその判定が記入され、添付されている。被験物質の添加量、測定値に関して適切性を確認するためのデータクリーニングが行われ、そのコメントも回収された。吸光度の転記ミスを防止するため 96 穴プレートの図示を用いたが、結果的には多くの記載ミスがあった。しかし確認後、適切に訂正され、意図的なデータの改変は認めなかった。
皮膚モデルの前培養時間についてのコメントはなく、結果的に影響は認めなかったが、SOP 等で変動幅がある項目についてはコメントを記録するべきである。
ピペットや電子天秤、マイクロプレートリーダー等の機器類は点検補正記録を保存しておくことがデータの質の向上につながると思われる。（GLP 適合施設は問題外）
9. 他の科学的な報告との比較の有無
OECD の in vitro 腐食性試験ガイドラインとして、「摘出皮膚電気抵抗性試験(TER)」、「ヒト皮膚モデ
ル試験」及び「in vitro 膜バリア試験」が承認されている。これらはいずれもバリデーション研究が実施され、ICCVAM はこれらの試験法(Rat Skin TER, EPISKIN, EpiDerm 及び Corrositex)の特異性、感度、正確性、偽陽性率及び偽陰性率について比較した結果を報告している。
本研究での結果はこれらと比較されている。試験物質の数量や選択物質の種類が異なっているため
結果の判定だけをもって、単純に皮膚モデルの優越性を評価することは困難であるが、Vitrolife-Skin™は少なくとも現在最も予測性の高いモデルとして確立されている EpiDerm™と同等の予測性を有すると思われる。
10. ３Rs への関与（動物福祉面からの妥当性）
いずれの皮膚モデルとも動物を使用しておらず、動物福祉面から代替法として妥当である。
11. 試験法の有用性と限界（コスト、時間からの妥当性など）
Vitrolife-Skin™1 キット(24 well)の価格は約 6 万円、1 試験で陽性、陰性対照とともに使用する場
合 8 well 使用で 3 試験が実施可能であり、1 試験当たりのコストは約 2 万円である。これは EpiDerm™
の約半分に相当する。試験実施に費やす時間は両試験とも 1 日以内であり、in vivo との一致率も同等と考えられた。
Vitrolife-Skin™は製品を入手後、直ちに前培養を施し実験を開始する必要がある。また、この前培
養時間の差異による結果への影響については明らかではない。Vitrolife-Skin™はポリカーボネート膜
上に表皮が構築された EpiDerm™よりヒトの皮膚構造に類似し、線維芽細胞を含むコラーゲン上に表皮層が重層化されている。しかし、被験物質が強アルカリ性の場合は基盤であるコラーゲンを溶解することにより、染色に必要な皮膚モデルの切り抜きが不可能となり測定に支障をきたした例があった。
しかし、この場合は腐食性物質と判定することが適切であり、SOP に対応する記載が必要であるとしている。同様に、被験物質によってはコラーゲンが膨張し、ポンチによる組織の切り取りが均一でない可能性がある。また、着色性物質やアルカリ性物質のような細胞や培養器材への吸着が強いものにつ10いては MTT の発色に影響を及ぼす可能性があり、コラーゲンのみのブランクを測定する等、配慮が必要と考えられる。これらの事項は Vitrolife-Skin™の改善点として挙げられている。
今後、大量生産等のため製造方法が変更される可能性がある。その場合、本研究に用いられたものと同質性を保証することが必要である。皮膚モデルの品質の同等性や反応性の均一化に関する規格を定めておく必要がある。
12. その他（特許の有無など）
特許については示されていない。Vitrolife-Skin™は既に市販されている。
13. 結論
Vitrolife-Skin™は皮膚腐食性評価のための皮膚モデルとして、EpiDerm™と同等の判定の正確性、再現性、信頼性を有していた。Vitrolife-Skin™を用いた皮膚腐食性試験は、動物や特殊な機器を使用せず短時間で容易に評価できたる。また、EpiDerm™に比べ安価であり、本邦で製造販売されているため安定した供給と管理が可能である。よって、Vitrolife-Skin™は皮膚腐食性試験の代替法として有用であると結論した。
14．文献
Barratt, M.D. et al., The ECVAM International Validation Study on in vitro tests for skincorrosivity. 1. Selection and Distribution of the test chemicals. Toxicology in Vitro, 12,471-482 (1998)
Botham, P.A. et al., A prevalidation study on in vitro skin corrosivity testing: The reportand recommendations of ECVAM workshop 6. ATLA, 23, 219-255 (1995)
Fentem, J.H. et al., The ECVAM International Validation Study on in vitro tests for skincorrosivity. 2. Results and evaluation by the management team. Toxicology in Vitro, 12,483-524 (1998)
ICCVAM (2002) NIH Publication No.02-4502. ICCVAM Evaluation of EPISKIN, and EpiDerm (EPI-200)
and rat skin transcutaneous electical resistance (TER) assay: in vitro test method forassessing dermal corrisivity potential of chemicals.
ICCVAM (1999) NIH Publication No.99-4495. Corrositex: An in vitro test method for assessing
dermal corrisivity potential of chemicals.
Liebsch, M., et al., The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing. ATLA, 28, 371-401 (2000)
OECD guideline for the testing of chemicals. Draft proposal for new guideline: 431, in vitro
Skin Corrosion: Human skin model test.
Worth, A.P. et al., An evaluation of the proposed OECD testing strategy for skin corrosion.
ATLA, 26, 709-720 (1998)
吉村功ら(2005)日本動物実験代替法学会バリデーション委員会、皮膚腐食性試験バリデーション結

果報告.

11

皮膚腐食性試験バリデーション結果報告

日本動物実験代替法学会バリデーション委員会
委員長 吉村 功

実行委員会委員
吉村 功（東京理科大学 工学研究科 経営工学）
大森 祟（京都大学大学院 医学研究科 社会健康医学系専攻
医療統計学部門）大野泰雄（国立医薬品食品衛生研究所 安全生物研 薬理部）
小島 肇（日本メナード化粧品(株) 総合研究所 研究技術部門
第二部安全性評価研究室）
実験責任者
安東朋子（国立医薬品食品衛生研究所 安全生物研 毒性部）
稲垣勝裕（日本農薬（株）総合研究所 安全性・医薬研究センター、
安全性グループ）
久保木真美（日本曹達（株）小田原研究所 安全性研究部）
小坂忠司（（財）残留農薬研究所 毒性第二部 免疫毒性研究室）
小島 肇（日本メナード化粧品(株) 総合研究所 研究技術部門
第二部安全性評価研究室）
中村洋介（住友化学工業（株）生物環境科学研究所生化学グループ）
皮膚三次元モデル提供者
鳥島 久（倉敷紡績（株）バイオメディカル部）
森川訓行（グンゼ（株）研究開発センター第５研究室）

1/7

序
動物福祉への関心の高まりに応ずるため、多くの動物実験代替法（以後、代替法と記す）が開発されてきた。それらの内、皮膚への直接作用としての強い障害性を調べる試験法の代替法（皮膚腐食性試験代替法）として、EUのECVAM Scientific Advisory Committeeは 1998 年にEPISKINTM とラット
Transcutaneous Electrical Resistance Test皮膚腐食性試験(TER法)を、2000 年にはEpiDermTM とCORROSITEXTM を認めた。米国では 1998 年にCORROSITEXTMを全ての化学物質に有用とは言えないが、Department of Transport（DOT）で必要とされる状況においては有用であると評価した1) 。OECDにおいても、TER法とEPISKINTM, EpiDermTM法が皮膚腐食性試験として専門家会議で承認され（それぞれガイ
ドライン 430 と 431）、正式な通知が待たれている。TER法を除き、これらの方法はいずれもヒト皮膚三次元モデルを利用した試験法である。しかし、これらの試験法について、我が国で検討された例が無く、手技や評価に関わる問題点が不明である。
我が国においてもVitrolife-SkinTM（グンゼ）やTESTSKINTM (東洋紡)のような皮膚三次元モデルが開発され、皮膚刺激性等を評価するために市販されているが、これらヒト皮膚三次元モデルを皮膚腐食性試験代替法として利用することについては検討された例がない。
一方、10%水酸化カリウムやフェノールのように、皮膚障害性の強い化学物質は劇物と分類されているが、in vivoで強い皮膚障害を起こすと予想される物質を実験動物を用いて検討することは、強い苦痛が予想されることから、動物福祉の立場から実施が困難である。我が国においても、強い皮膚障害性の判定に、上記のin vitro皮膚腐食性試験を利用することが望ましい。
以上の状況を鑑み、日本でヒト皮膚三次元モデルとして開発されたVitrolife-SkinTMとすでにECVAM
にてバリデーションが終了しているEpiDermTMの特性を明らかにするために、多施設での小規模バリデーションを実施した。

方法
１） バリデーション組織
わが国で皮膚三次元モデルを販売している企業に基本計画を示し、バリデーションへの協力を求めたところ、クラボウ社（EpiDermTM）およびグンゼ社（Vitrolife-SkinTM）より協力が得られた。そこで、
日本動物実験代替法学会及び厚生労働省医薬局生活科学安全対策室を通じて、バリデーションへの参加施設を募集した。その結果、国立医薬品食品衛生研究所（毒性部）、日本農薬、日本曹達、残留農薬研究所、日本メナード化粧品及び住友化学工業より参加の申し入れがあった。そこで、日本動物実験
代替法学会にバリデーションへの協力を求め、そのバリデーション委員会を中心に少数の被験物質を用いたバリデーション研究を実施するとされ、バリデーション実行委員会を組織した（添付資料１）。
２） 被験物質および器材
バリデーション実行委員会では今までの皮膚腐食性試験のバリデーションで用いられた被験物質とその腐食性を調査した（添付資料 2）。実行委員会ではそれらの中から予備試験及び本試験用の陽性対照物質として 10%水酸化カリウム水溶液、20% Sodium lauryl sulfate 水溶液および塩化ベンザルコニ
2/7ウムを、また、実験に直接関与しない国立医薬品食品衛生研究所薬理部の大野が被験物質として 12 物質を選考した（表 1）。被験物質は大野がコード化し、実験実施施設に配布した。各施設には陽性対照
物質とともに、腐食性物質と非腐食性物質をそれぞれ 5 物質ずつ配布した（表 2）。なお、配布された
被験物質はすべて劇物として扱うこと、冷暗所に保管すること、また、GLP に準じて、使用・保管記録をとるよう指示された。
ヒト皮膚三次元モデルEpiDermおよびVitrolife-SkinTMはそれぞれクラボウ社とグンゼ社より購入し、実験スケジュールに従い、実験施設に直接配布した。配布数は予備試験を１回及び本試験を２回とし、それぞれ 2 キットずつ使用することから、6 キット配布するとしたが、追加試験を行う場合は、追加のキットを配布した。
３）バリデーションの試験計画書の作成と技術移転
バリデーション実行委員会ではバリデーション実施に際しての詳細を検討し、試験計画書（添付資料 3）および標準操作手順書（SOP, 添付資料 4）を作成し、それに基づいて、陽性対照物質を用いて技術移転を行った。
４）バリデーション実験の実施
適正な技術を持った者により実験が行われるよう、三次元モデルを用いた実験を行う者は技術移転の会に出席した参加施設のうち、本試験開始までにキットを使用した経験のある試験担当者に限るとした。試験期間は平成 16 年 2〜3 月とした。
なお、実験は予備試験を１回、本試験を 2 回行い、2 回の本試験で最終判定が食い違った物質、および技術的な問題で１例のみしか結果が得られなかった物質については、追加試験を行うこととした。
なお、追加試験のためのキットを予め注文してあったため、2 回の本試験で同じ結果が得られた場合においても確認のため、追加試験を実施した場合もある。
５）腐食性の判定
被験物質の腐食性の最終判定は、2〜3 回の本試験結果のうち、同一の判定の得られた 2 例の結果を最終判定結果とすることとした。しかし、追加試験のためのキット提供の都合から、4 月末に実験が終了した。
試験の実施に際しては GLP の原則を尊重し、試験計画書および SOP に従って実施された。また、試験記録および修正記録を明確にとることとされた。大きな逸脱があった場合には、結果を採用しないこともあるとされた。
６）データ解析
試験結果は実行委員長の吉村に提供するとされた。そこで、すべてのデータが提出されたことが確認されたところで、大野より被験物質コードと参加施設への割付表が開示された。これに基づいて、吉村がデータを確認したのち、結果を固定し、解析した。

結果
それらの結果を表 3 および 4 にまとめた。
１） 試験結果の再現性
3/7

2 回の本試験で異なる結果が出たのは、EpiDermTMにおいては、66 試験の内sulfuric acid、Lactic acid
で各 2 施設であり、発生率 4/66、即ち、6.06%であった。Vitrolife-SkinTMにおいては、sulfuric acid、
Octanoic acid、Sodium hydroxide(4.88％)で各１施設であり、発生率 3/66、即ち、4.55%であった。
２） 予知能力2 回の試験で異なる結果が得られた場合の判定については、３回目の試験を実施し、その結果を合わ
せ、3 回の試験結果を基に多数決で判定するとされている。そこで、これに従って判定された結果を基に、被験物質の腐食性を判定した。
EpiDermTMで腐食性物質を正しく腐食性と判定した事例は 6 被験物質 30 試験中、29 試験であり、感度(sensitivity)は 29/30、即ち、96.6%であった。一方、非腐蝕性物質を正しく非腐蝕性と判定した事例
は 6 被験物質 30 試験中、20 試験であり、特異性(specificity)は 20/30、即ち 66.7%であった。なお、
非腐食性物質を誤って腐食性物質と判定したのは、5% Potassium hydroxideとLactic acidであり、この場合、いずれの施設でも腐食性物質と判定した。腐食性との結果が得られたもののうち，真に腐食性である割合、陽性予知能力(positive predictivity)は 29/39 即ち、74.4%であった。非腐食性との結果が得られたもののうち、真に非腐食性である割合、陰性予知能力(negative predictivity)は 20/21、
即ち、95.2%であった。正しい判定結果が得られた割合、一致率は 49/60 即ち、81.7%であった。
Vitrolife-SkinTMでは感度は 30/30、即ち 100%であり、特異性は 20/30、即ち 66.7%、陽性予知能力は 30/40、即ち 75%、陰性予知能力は 20/20、即ち 100%、一致率は 50/60、即ち 83.3%であった。EpiDermTMの場合と同様に、非腐食性物質を誤って腐食性物質と判定したのは、5% Potassium hydroxideとLactic
acidであり、いずれの施設でも腐食性物質と判定した。
これらのように、いずれの場合も、EpiDermTMとVitrolife-SkinTMによりほとんど同じ結果が得られた。
また、判断を誤る物質も同じであった。

考察
OECDやEU, 米国で皮膚腐食性試験代替法として承認されているEpiDermTM と我が国で開発されたVitrolife-SkinTMの 2 種のヒト皮膚三次元モデルについて、OECDのバリデーション基準に準じて、多施設バリデーションを実施した。その結果、前者ではfalse negativeが 30 試験のうち 1 試験で認められたが、後者ではfalse negativeは認められなかった。一方、両皮膚三次元モデルの試験法とも 5% 水酸化カリウムと乳酸でfalse positiveとなった。結果としては、false negative及びfalse positiveの発現については，両皮膚三次元モデルともほとんど同じ結果が得られた。
今回、使用した被験物質数は陽性対照物質を除き、12 物質であり、従来のバリデーションと比べ、少ないが、OECD で適切なガイドラインとして専門家会議で承認されている。皮膚三次元モデルと同等の も ので あり 、 完全 に新 規 の方 法と 比 べて より 小 数の 被験 物 質で のバ リ デー ショ ン (catch up
validation)でその妥当性を判断できると考えたものである。
一方、水酸化カリウム、硫酸、水酸化ナトリウム、及びフェノールは我が国の毒劇物判定において、皮膚粘膜刺激性において、これと同等以上の物質は劇物と判定するとされているものであるが、それぞれ 10%硫酸, 5%水酸化ナトリウム, 5%水酸化カリウム、及び 5%フェノールと同等以下の刺激性を有4/7

する物質については劇物から除外されているものである。一方、5%水酸化カリウムを除き、10%硫酸や5%水酸化ナトリウムは腐食性物質として報告されているものであり、今回の試験においても腐食性との結果が得られた。これらを考えると、本試験を日本で採用する場合においては、皮膚粘膜刺激性の観点からの劇物の判定基準を見直す必要があると考えられる。

Vitrolife-SkinTMにおいて、強度のアルカリにより本皮膚三次元モデルの支持組織であるコラーゲン
スポンジが完全に溶けてしまい、腐食性の判定に支障を来した。今回の判定においては、強い刺激性があるものとして、腐食性と判定したが、SOP上もそのように改定する必要がある。なお、酸やアルカ
リ検体については、その他の障害は認められておらず、pH変化によるviability測定のための色素の発色への影響は洗浄などの操作により解消できたと考えられる。一方、着色物質の中には、細胞や培養基材への吸着の強いものがあることも考えられることから、それら被験物質の場合には取り扱いには注意する必要がある。また、ポンチで切り取る組織の大きさが皮膚モデルの膨潤率により変化する可能性があり、注意を喚起する必要があるとされた。更に、Vitrolife-SkinTMでは被験物質として用いたアルカリがコラーゲンスポンジに残留することにより発色が影響される場合があり、このような被験物質の場合ではコラーゲンスポンジのみのブランクが必要であった。
引用文献
1) ICCVAM: CorrositexR: An in vitro test method for assessing dermal corrosivity potential ofchemicals. The results of an independent peer review evaluation coordinated by theInteragency Coordinating Committee on the validation of Alternative Methods (ICCVAM) andthe National Toxicology Program (NTP) interagency center for the evaluation of alternative
toxicological methods (NICEATM). NIH Publication No: 99-4495 (1999).

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表１：被験物質
番号

被験物質名

腐食性*

コメント

1

Potassium hydroxide(10%)

C

陽性対照物質劇物

液体2

Sodium lauryl sulfate(20% )NC

陽性対照物質液体

3

Sulfuric acid (10%)

C

劇物判定薬

非劇物 液体4Octanoic (Caprylic) acidC

5

Sodium hydroxide(4.88%)

C

劇物判定薬

非劇物 液体6

Phenol

C

劇物判定薬

劇物

固体 mp 40-43℃

7

Chromiun trioxideC

劇物

固体 mp 197℃

8

Phosphoric acidC

非劇物 固体 mp 42.3℃

9Sodium perborateNC

非劇物 粉末 60℃で分解

10 TetrachloroethyleneNC

非劇物 液体 bp 121℃

11 Potassium hydroxide (5%)

NC

液体

12 4-Amino-1,2,4-triazoleNC

非劇物 粉末 mp159℃

13 L-Lactic acidNC

非劇物 固体 mp 52.8℃

14 Isopropanol (2propanol)
C: corrosive, NC: non-corrosiveNC

非劇物 液体 bp 82.5℃

非劇物 液体 bp 239.7℃

表２：被験物質の各施設への配布表
被験物質名

腐食性* 施設１

施設２

施設３

施設４

施設５

施設６

Potassium hydroxide (10%)

C

1

2

3

4

5

6

Sodium lauryl sulfate (20%)

NC

7

8

9

10

11

12

Sulfuric acid (10%)

C

13

14

15

16

17

×

Tetrachloroethylene

NC

18

19

20

21

×

22

Octanoic (Caprylic) acid

C

23

24

25

26

×

27

Potassium hydroxide (5%)

NC

28

29

30

×

31

32

Sodium hydroxide (4.88%)

C

33

34

35

×

36

37

4-Amino-1,2,4-triazole

NC

38

39

×

40

41

42

Phosphoric acid

C

×

43

44

45

46

47

L-Lactic acid

NC

48

×

49

50

51

52

Isopropanol (2-propanol)

NC

×

53

54

55

56

57

Phenol

C

58

59

×

60

61

62

Sodium perborate

NC

63

64

65

66

67

×

Chromiun trioxide

C

68

×

69

70

71

72

C: corrosive, NC: non-corrosive

6/7Unclassified

ENV/JM/MONO(2005)14

Organisation de Coopération et de Développement Economiques
Organisation for Economic Co-operation and Development

18-Aug-2005________________________________________________________________________________________________________English - Or. English
ENVIRONMENT DIRECTORATEENV/JM/MONO(2005)14
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYOECD SERIES ON TESTING AND ASSESSMENT
Number 34
GUIDANCE DOCUMENT ON THE VALIDATION AND INTERNATIONAL ACCEPTANCE OF NEW
OR UPDATED TEST METHODS FOR HAZARD ASSESSMENT

Patric AMCOFF
Tel: +33 (0)1 45 24 16 19; Fax: +33 (0)1 44 30 61 80; Email: patric.amcoff@oecd.org
English - Or. English

JT00188291

Document complet disponible sur OLIS dans son format d'origine
Complete document available on OLIS in its original format

ENV/JM/MONO(2005)14

OECD Environment, Health and Safety Publications
Series on Testing and Assessment
No. 34

GUIDANCE DOCUMENT ON THE VALIDATION AND
INTERNATIONAL ACCEPTANCE OF NEW OR UPDATED TEST METHODSFOR HAZARD ASSESSMENT

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENTParis
18 August 2005

2

ENV/JM/MONO(2005)14
Also published in the Series on Testing and Assessment:

No. 1, Guidance Document for the Development of OECD
Guidelines for Testing of Chemicals (1993; reformatted 1995)
No. 2,

Detailed Review Paper on Biodegradability Testing (1995)

No. 3,(1995)

Guidance Document for Aquatic Effects Assessment

No. 4, Report of the OECD Workshop on Environmental
Hazard/Risk Assessment (1995)
No. 5, Report of the SETAC/OECD Workshop on Avian
Toxicity Testing (1996)
No. 6, Report of the Final Ring-test of the Daphnia magna
Reproduction Test (1997)
No. 7, Guidance Document on Direct Phototransformation ofChemicals in Water (1997)
No. 8, Report of the OECD Workshop on Sharing Information
about New Industrial Chemicals Assessment (1997)
No. 9, Guidance Document for the Conduct of Studies ofOccupational Exposure to Pesticides during Agricultural
Application (1997)
No. 10, Report of the OECD Workshop on Statistical Analysis of
Aquatic Toxicity Data (1998)
No. 11, Detailed Review Paper on Aquatic Testing Methods for
Pesticides and industrial Chemicals (1998)
No. 12, Detailed Review Document on Classification Systems forGerm Cell Mutagenicity in OECD Member Countries (1998)
No. 13, Detailed Review Document on Classification Systems for
Sensitising Substances in OECD Member Countries 1998)
No. 14, Detailed Review Document on Classification Systems
for Eye Irritation/Corrosion in OECD Member Countries (1998)
No. 15, Detailed Review Document on Classification Systems
for Reproductive Toxicity in OECD Member Countries (1998)
No. 16, Detailed Review Document on Classification Systems
for Skin Irritation/Corrosion in OECD Member Countries (1998)

3

ENV/JM/MONO(2005)14
No. 17, Environmental Exposure Assessment Strategies for
Existing Industrial Chemicals in OECD Member Countries(1999)
No. 18, Report of the OECD Workshop on Improving the Use of
Monitoring Data in the Exposure Assessment of Industrial
Chemicals (2000)
No. 19, Draft Guidance Document on the Recognition,
Assessment and Use of Clinical Signs as Humane Endpoints forExperimental Animals used in Safety Evaluation (1999)
No. 20, Guidance Document for Neurotoxicity Testing (2004)
No. 21, Detailed Review Paper: Appraisal of Test Methods for
Sex Hormone Disrupting Chemicals (2000)
No. 22, Guidance Document for the Performance of Out-door
Monolith Lysimeter Studies (2000)
No. 23, Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures (2000)
No. 24, Guidance Document on Acute Oral Toxicity Testing(2001)
No. 25, Detailed Review Document on Hazard Classification
Systems for Specifics Target Organ Systemic Toxicity Repeated
Exposure in OECD Member Countries (2001)
No. 26, Revised Analysis of Responses Received from Member
Countries to the Questionnaire on Regulatory Acute Toxicity
Data Needs (2001)
No. 27, Guidance Document On the Use of the Harmonised
System for the Classification of Chemicals Which Are Hazardous
For the Aquatic Environment (2001)
No. 28, Guidance Document for the Conduct of Skin Absorption
Studies (2004)
No. 29, Draft
Guidance
Documenton
Transformation/Dissolution of Metals and Metal Compounds in
Aqueous Media (2001)
No. 30, Detailed Review Document on Hazard Classification
Systems for Mixtures (2001)
No. 31, Detailed Review Paper on Cell Transformation Assays
for Detection of Chemical Carcinogens (draft)4ENV/JM/MONO(2005)14
No. 32, Guidance Notes for Analysis and Evaluation of RepeatDose Toxicity Studies (2000)
No. 33, Harmonised Integrated Classification System for Human
Health and Environmental Hazards of Chemical Substances and
Mixtures (2001)

No. 34, Guidance Document on the Validation and International
Acceptance of New or Updated Test Methods for Hazard
Assessment (2005)
No. 35, Guidance Notes for Analysis and Evaluation of Chronic
Toxicity and Carcinogenicity Studies (2002)
No. 36, Report of the OECD/UNEP Workshop on the use ofMultimedia Models for estimating overall Environmental
Persistence and long range Transport in the context of
PBTS/POPS Assessment (2002)
No. 37, Detailed Review Document on Classification Systems for
Substances which Pose an Aspiration Hazard (2002)
No. 38, Detailed Background Review of the Uterotrophic Assay
Summary of the Available Literature in Support of the Project of
the OECD Task Force on Endocrine Disruptors Testing andAssessment (EDTA) to Standardise and Validate the Utertrophic
Assay (2003)
No. 39, Guidance Document on Acute Inhalation Toxicity Testing(in preparation).
No. 40, Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures which cause
Respiratory Tract Irritation and Corrosion (2003)
No. 41, Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures which in contact
with Water release Toxic Gases (2003)
No. 42, Guidance document on Reporting Summary Information
on Environmental, Occupational and Consumer Exposure (2003)
No. 43, Guidance Document on Reproductive Toxicity Testingand Assessment (draft)
No. 44, Descriptions of Selected Key Generic Terms used in
Chemical Hazard/Risk Assessment (2003)
No. 45, Guidance Document on the Use of Mulimedia Models for
Estimating Overall Environmental Persistance and Long-Range
Transport (2004)
5

ENV/JM/MONO(2005)14

No. 46, Detailed Review Paper on Amphibian Metamorphosis
Assay for the Detection of Thyroid Active Substances (2004)
No. 47, Detailed Review Paper on Fish Screening Assays for the
Detection of Endocrine Active Substances (2004)
No. 49, Report from the Expert group on (Quantitative)
Structure-Activity Relationships [(Q)SARs] on the Principles for
the Validation of (Q)SARs (2004)
No. 50, Report from the OECD/ICPS
Toxicogenomics, Kyoto, October 2004 (2005)

Workshopon© OECD 2005 Applications for permission to reproduce ortranslate all or part of this material should be made to: Head of
Publications Service, OECD, 2 rue André-Pascal, 75775 Paris,
Cedex 16, France.

6

ENV/JM/MONO(2005)14

About the OECDThe Organisation for Economic Co-operation and Development (OECD) is an intergovernmentalorganisation in which representatives of 30 industrialised countries in North America, Europe and thePacific, as well as the European Commission, meet to co-ordinate and harmonise policies, discuss issues ofmutual concern, and work together to respond to international problems. Most of the OECD’s work is
carried out by more than 200 specialised committees and subsidiary groups composed of member country
delegates. Observers from several countries with special status at the OECD, and from interested
international organisations, attend many of the OECD’s workshops and other meetings. Committees andsubsidiary groups are served by the OECD Secretariat, located in Paris, France, which is organised into
directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in nine different
series: Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides
and Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety ofNovel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; andEmission Scenario Documents. More information about the Environment, Health and Safety Programme
and EHS publications is available on the OECD’s World Wide Web site (http://www.oecd.org/ehs/).

This publication was produced within the framework of the Inter-Organisation Programme for theSound Management of Chemicals (IOMC).

The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) wasestablished in 1995 following recommendations made by the 1992 UN Conference on
Environment and Development to strengthen co-operation and increase international coordination in the field of chemical safety. The participating organisations are FAO, ILO,
OECD, UNEP, UNIDO, UNITAR and WHO. The World Bank and UNDP are observers. The
purpose of the IOMC is to promote co-ordination of the policies and activities pursued by theParticipating Organisations, jointly or separately, to achieve the sound management ofchemicals in relation to human health and the environment.

7

ENV/JM/MONO(2005)14

This publication is available electronically, at no charge.
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)

or contact:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
FranceFax: (33-1) 45 24 16 75
E-mail: ehscont@oecd.org

8ENV/JM/MONO(2005)14
PREAMBLE
1.
The development of this Guidance Document started in 1998 as a follow-up to the 1996 Solna
Workshop on “Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test
Methods”. Whereas the principles and criteria for validation and regulatory acceptance of new and revised
test methods, agreed in Solna and included in the report of the Workshop (1), were widely accepted, it
appeared that there was still a need to expand on these principles and provide more guidance on validationin general. Moreover, practical experience with validation studies has substantially accumulated since theSolna Workshop, and using this experience to provide more specific guidance was considered very useful.
2.
A crucial step in the initial development of this Guidance Document was the OECD Secretariat's
consultation with a selected number of internationally recognised experts in the area of test validation. The
contributions from these experts, both in terms of practical advice, suggestions and final development of
the first draft Guidance Document on “The Development, Validation and Regulatory Acceptance of New
and Updated Test Methods in Hazard Assessment" have been extremely helpful. The draft GuidanceDocument was circulated to Member countries and other stakeholders in late 2001 and the OECD
Secretariat received comments from eight member countries in addition to comments from EC, ICAPO andBIAC.
3.
An OECD Conference on “Validation and Regulatory Acceptance of New and Updated Methods inHazard Assessment” was held in Stockholm, Sweden in March 2002. The stated purpose of the conference
was to develop, and achieve consensus on, practical guidance on principles and processes for the validation
and acceptance of animal and non-animal test methods for regulatory hazard assessment purposes. Thisconsensus guidance would be used to revise and further improve the draft OECD Guidance Document. The
Stockholm Conference agreed upon 40 specific recommendations on how to improve and extend the draft
Guidance Document (2). In addition the Conference agreed upon a number of general suggestions andeditorial corrections for the upcoming revision taking into consideration the comments received after the
first circulation round of the draft Guidance Document.
4.
Two of the most controversial areas that many participants of the Conference felt were not properly
covered were the issues of Data Interpretation Procedures (DIP) and the Use of Human Data. Therefore,
the Conference made a strong recommendation that two separate OECD Workshops should be organised tofurther examine these issues. With the purpose of speeding up the finalisation of the draft GuidanceDocument, the Conference decided that a small Drafting Group should be established as soon as possible
after the Meeting to initiate the revision. The group comprised representatives from the EU, Japan andNorth American Regulatory Communities as well as representatives from BIAC and ICAPO. In December
2002, the Secretariat established the Drafting Group through an official nomination round and in total eight
experts offered their assistance. The Drafting Group had its first telephone conference in February 2003,
during which the outline and approach of the revision was discussed and several topics that deserved extra
attention were identified. These so called “hot-spot issues” were: (i) validation of (Q)SARs; (ii), Data
Interpretation Procedures (DIP); (iii), the use of human and existing data; (iv), distinctions between
different phases of the validation process; (v), retrospective validation and validation by convention; and,(vi) development of Test Guidelines from validated protocols.
5.
The members of the Drafting Group came together in March 2003 in Paris to start the revision of
the draft Guidance Document. The Drafting Group discussed and agreed on how to organise the work
taking into account the outcome from the Stockholm Conference and the previous commenting round of
the draft. The revised 2nd version was circulated to the WNT for comments in October 2003, and again, aconsiderable number of comments were received. Given the scope and extent of the comments, theSecretariat asked the WNT16 for guidance on how to continue the revision of the document. The US
offered to host an Expert Consultation meeting in Bethesda, Maryland, to resolve these last topics. The
9

ENV/JM/MONO(2005)14
meeting was held on October 13-15, 2004, and successfully addressed the comments received in addition
to making a number of rather extensive amendments to the draft Guidance Document.
6.
One of the recommended follow-up activities of the Stockholm Conference was a workshop on
DIPs, and after a kind offer from Germany (ZEBET) a workshop was held in Berlin on 1-2 July, 2004. The
main purpose being to try to achieve consensus on principles for validation, and use of DIPs and Prediction
Models (PMs) that are applicable to both in vivo, in vitro and ecotoxicological test methods. The workshop
discussed and agreed upon a number of items, including terminology, and provided further guidance forthe revision of draft Guidance Document 34. However, a consensus on the applicability of DIPs and PMs
for different types of methods was not achieved. A Meeting Report is available (3).
7.
Many experts have participated in the OECD meetings since the Solna Workshop in 1996 and theSecretariat would especially like to mention the experts that have been actively involved in thedevelopment and drafting of this document, including:– Hans Ahr, Bayer AG, Wuppertal, Germany– Michael Balls, EC/ECVAM, Ispra, Italy– Robert Boethling, US-EPA, Washington, DC, USA– Dorothy Canter, US-EPA, Washington, DC, USA– Mark Chamberlain (BIAC) Unilever, UK– Alan Goldberg, CAAT, Baltimore, MD, USA– Petra Greiner, UBA, Germany– Kailash Gupta, CPSC, Bethesda, MD, USA– Karen Hamernik, US-EPA, Washington, DC, USA– David Hattan, FDA, Bethesda, MD, USA– Abigail Jacobs, FDA, Rockville, MD, USA– Manfred Liebsch, ZEBET, Berlin, Germany– Kimmo Louekari, Product Control Agency for Welfare and Health , Finland– Yasuo Ohno, NIHS, Tokyo, Japan– Willie Owens, (BIAC) Procter and Gamble, USA– Richard Phillips, Exxon/Mobile, East Millstone, NJ, USA– Amy Rispin, US-EPA, Washington, DC, USA– Andrew Rowan, Humane Society of the US, Washington, DC, USA– Len Schechtman, FDA, Rockville, MD, USA– Jerry Smrchek, US-EPA, Washington, DC, USA– Horst Spielmann, ZEBET, Berlin, Germany– Martin Stephens (ICAPO), USA– William Stokes, NIEHS, Research Triangle Park, NC, USA– Gary Timm, US-EPA, Washington, DC, USA– Leslie Touart, US-EPA, Washington, DC, USA– Neil Wilcox, formerly of FDA, Rockville, MD, USA– Marilyn Wind, CPSC, Bethesda, MD, USA– Andrew Worth, JRC-EC, Italy– Errol Zeiger, formerly of NIEHS, Research Triangle Park, NC, USA
The OECD Secretariat gratefully acknowledges these experts and others who have contributed to theproject for their professional assistance and their indispensable contributions to the finalisation of thisimportant Guidance Document.
8.
This Guidance Document should be considered in conjunction with Guidance Document No.1
(Development of OECD Guidelines for Testing of Chemicals (4)) published in this same series of
Guidance Documents on Testing and Assessment.
10ENV/JM/MONO(2005)14
TABLE OF CONTENTS
PREAMBLE ....................................................................................................................................................9I. INTRODUCTION......................................................................................................................................13
II. DEFINITION OF THE TEST METHOD.................................................................................................17
III. APPROACHES TO VALIDATION .......................................................................................................20
Prospective Validation Studies ......................................................................................................................20
Retrospective Assessment of Validation Status.............................................................................................21“Modular” Approaches to Assessing Validation Status ................................................................................22
Performance Standards for Test Methods......................................................................................................24
Use of Performance Standards for Catch-up Validation................................................................................24
Use of Performance Standards for Modified Test Methods ..........................................................................25
Validation of Patented Methods.....................................................................................................................25
Validation of Test Batteries/Testing Strategies .............................................................................................25
Validation of (Quantitative) Structure-Activity Relationships - (Q)SARs ....................................................26
IV DESIGN AND CONDUCT OF VALIDATION STUDIES ....................................................................27
General Considerations..................................................................................................................................27
Management of Validation Studies................................................................................................................31
Statistical Expertise........................................................................................................................................31
Conduct of the Prevalidation .........................................................................................................................32
Project Plan....................................................................................................................................................34
Participating Laboratories..............................................................................................................................34
Reference Data...............................................................................................................................................35
Selection of Test Chemicals and Chemical Management..............................................................................36
Coding and Distribution of Test Samples......................................................................................................38
Test Method Decision Criteria and Data Interpretation.................................................................................39
Monitoring of Participating Laboratory Performance ...................................................................................39
Inter-laboratory Testing .................................................................................................................................40
Data Collection ..............................................................................................................................................40
Data Analysis.................................................................................................................................................41
Reporting .......................................................................................................................................................42
Record-Keeping/Data Dissemination ............................................................................................................42
V. INDEPENDENT EVALUATION OF A VALIDATION STUDY (PEER REVIEW)............................43
Mechanisms for Peer Review ........................................................................................................................43
Selection of Peer Reviewers ..........................................................................................................................44
Charge to Peer Reviewers..............................................................................................................................45
Peer Review Process......................................................................................................................................45
VI. INTERNATIONAL REGULATORY ACCEPTANCE OF VALIDATED TESTS...............................47
Validation Study Outcomes ...........................................................................................................................47
Criteria for Regulatory Acceptance ...............................................................................................................47
From Protocol to Test Guideline....................................................................................................................51
VII. NEW TEST SUBMISSIONS: SUPPORTING DOCUMENTATION ..................................................50
Introduction and Rationale for the Proposed Test Method ............................................................................50
Test Method Protocol Components ...............................................................................................................50
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ENV/JM/MONO(2005)14
Characterisation and Selection of Substances Used for Validation of the Proposed Test Method................51
In Vivo Reference Data Used to Access the Accuracy of the Proposed Test Method ...................................51
Test Method Data and Results .......................................................................................................................53
Test Method Relevance (Accuracy)...............................................................................................................53
Test Method Reliability (Repeatability/Reproducibility) ..............................................................................54
Test Method Data Quality..............................................................................................................................54
Other Scientific Reports and Reviews ...........................................................................................................54
Animal Welfare Considerations (Refinement, Reduction and Replacement) ..............................................54
Practical Considerations ................................................................................................................................55
References......................................................................................................................................................55
Supporting Materials......................................................................................................................................55
VIII. REFERENCES .....................................................................................................................................56
ANNEX I: Definitions and Glossary .............................................................................................................62
ANNEX II: Examples of validation studies within different areas of test method development ..................69
Example 1: The OECD Lemna growth inhibition test. Development and ring-testing ofDraft OECD test Guideline............................................................................................................................72
Example 2: Report of the Final Ring Test of the Daphnia magna Reproduction Test..................................75
Example 3: Validation, Special Study and “catch-up” Validation of In vitro Tests for Skin............................
Corrosion Potential – The Human Skin Model Test......................................................................................79
Example 4: The Local Lymph Node Assay (LLNA).....................................................................................86
Example 5: The Up-and-Down Procedure for Acute Oral Toxicity (TG 425) ..............................................93
TABLES AND FIGURES
Table 1: Principles and criteria for test method validation. ........................................................................ 23
Figure 1: From test development to validation: entry points for test method optimisation ........................ 28
Figure 2: Key factors for validation and regulatory acceptance of new and revised toxicology
test methods ................................................................................................................................................ 29
Figure 3: Role and interactions of the validation manager/management team........................................... 33
Table 2: Principles and criteria for regulatory acceptance of a new test method. ...................................... 48
Table 3: Characterisation of chemicals tested. ........................................................................................... 51
Table 4: Test method accuracy assessment. ............................................................................................... 53

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ENV/JM/MONO(2005)14

I. INTRODUCTION
9.
Test Guidelines, including those produced by the OECD, are used by governments, industry andindependent laboratories to determine the hazard or safety of chemicals. The use of Test Guidelines thatare based on validated test methods promotes the generation of dependable data for human and animal
health and environmental hazard assessment. It has been recognized that there is a need to develop
guidance for the international community that outlines general principles, important considerations,
illustrative examples, potential challenges and the results of experience gained in the area of test method
validation. This guidance document has been drafted by representatives of OECD Member countries,
based on advice from Member countries and OECD stakeholders.
10.
The purpose of this document is to provide guidance on issues related to the validation of new or
updated test methods consistent with current approaches. This document provides a synopsis of the current
state of test method validation, in what is a rapidly changing and evolving area. Guidance on the moregeneral aspects of OECD Test Guideline development is provided in the OECD Guidance Document forthe development of OECD Test Guidelines for the testing of chemicals (4).While the principles ofvalidation as described in this document were written for biology-based tests, they may be applicable toother Test Methods.
11.
Test method validation is a process based on scientifically sound principles (5)(6) by which thereliability and relevance of a particular test, approach, method, or process are established for a specificpurpose. Reliability is defined as the extent of reproducibility of results from a test within and amonglaboratories over time, when performed using the same standardised protocol. The relevance of a test
method describes the relationship between the test and the effect in the target species and whether the test
method is meaningful and useful for a defined purpose, with the limitations identified. In brief, it is theextent to which the test method correctly measures or predicts the (biological) effect of interest, as
appropriate. Regulatory need, usefulness and limitations of the test method are aspects of its relevance.
New and updated test methods need to be both reliable and relevant, i.e., validated.
12.
A set of principles for validation, also called the “Solna Principles”, were developed at an OECD
Workshop in Solna Sweden in 1996, where it was agreed that these Principles apply to the validation of
new or updated test methods for hazard assessment, whether they are in vivo or in vitro, or tests for effects
on human health or the environment (1). These Principles are:
1) A rationale for the test method should be available. This should include a clear statementof scientific need and regulatory purpose.
2) The relationship of the endpoint(s) determined by the test method to the in vivo biologicaleffect and to the toxicity of interest should be addressed. The limitations of a methodshould be described, e.g., metabolic capability.
3) A formal detailed protocol must be provided and should be readily available in the publicdomain. It should be sufficiently detailed to enable the user to adhere to it, and it should
include data analysis and decision criteria. Test methods and results should be available
preferably in an independent peer reviewed publication. In addition, the result of the test
should have been subjected to independent scientific review.
4) Intra-test variability, repeatability and reproducibility of the test method within andamongst laboratories should have been demonstrated. Data should be provided describing
the level of inter- and intra-laboratory variability and how these vary with time.
5) The test method’s performance must have been demonstrated using a series of reference
chemicals preferably coded to exclude bias.
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ENV/JM/MONO(2005)14
6) The performance of test methods should have been evaluated in relation to existing
relevant toxicity data as well as information from the relevant target species.
7) All data supporting the assessment of the validity of the test methods including the full
data set collected in the validation study must be available for review.
8) Normally, these data should have been obtained in accordance with the OECD Principles
of Good Laboratory Practice (GLP).
13.
Given the continuing increase in the numbers and types of test methods being developed forvarying purposes, the validation process should be flexible and adaptable. The extent to which thesevalidation principles are addressed will vary with the purpose, nature, and proposed use of the test method.
There are differences between in vivo assays and in vitro or ex vivo assays which should be considered inapplying the validation principles. (7) In general, the closer the linkage between the effect measured andthe toxicological effect of interest, the easier it will be to establish the relevance of the assay. The moreclosely a test measures/observes (an) effect(s) identical to the effect(s) of concern, the greater theconfidence that the test will accurately characterize or model the effect in the target species of concern. (8)14.
There have been significant advances in test method development for toxicology andecotoxicology over time. Many toxicity and ecotoxicity test methods have been in routine use for many
years and have yielded, and continue to yield, information relevant to the toxicity of chemicals. Regulatory
authorities and the regulated community accept these test methods for hazard and risk assessment purposes
without the prerequisite for formal validation of those test methods using present-day validation criteria.
OECD Test Guidelines are largely based upon such test methods.
15.
Many alternative tests for potential human health hazards have been subjected to collaborative
validation studies that preceded the systematic harmonization of validation principles by national or supranational organisations/agencies (i.e., ICCVAM, the Interagency Coordinating Committee on the Validationof Alternative Methods and ECVAM, the European Centre for the Validation of Alternative Methods).
These test methods are accepted by international and national testing organizations and regulatory
authorities for hazard and risk assessment based upon their history of use and proven utility. They provide
a strong scientifically-based foundation for regulatory decision-making.
16.
A number of these human health effect test methods are now used as the standards against which
newly developed alternative test methods are evaluated. The ecotoxicological community has also
conducted validation studies (often referred to as “ring-tests”) for decades to determine the reliability oftest methods. Their focus has been on standardization of protocols and demonstrations of theirtransferability and reproducibility.
17.
New test methods undergo validation to assure that they employ sound science and meet regulatoryneeds. Such new test methods have been developed for reasons that include human and ecological healthconcern, animal welfare considerations, data quality assurance, and to incorporate new and improved
technologies. Increasing concern about the use of laboratory animals for toxicity studies and other effectsof substances has led to widespread support of, and adherence to, the principle of the 3Rs of animal use inalternative test method development [Replacement, Reduction and Refinement (9)]. Regulatory authorities
have endorsed the principle of the 3Rs. As a consequence, alternative test methods have been developed toreplace the use of animals with non-animal systems, reduce the number of animals in a test, or refine theprocedures to make them less painful or stressful to the animals under study (10).
18.
With the introduction of new test methods, emphasis on continued test development and the need
to work towards international acceptance of new or updated test methods, it has become apparent that a

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means to promote international awareness of general principles, issues, and examples related to thevalidation of new test methods is needed.
19.
This document provides guidance on the conduct of validation studies and how to demonstrate thatthe test methods have been appropriately validated. Validation studies can range from small-scale activities
to large-scale multi-national programmes. Studies to support or effect validation can be performed under
the auspices of the OECD, by individual Member countries, by test method sponsors, or by a number ofdifferent organisations. For example, some organisations are specialized in running test method validationof alternative test methods and/or are established by law as validation institutions, such as ECVAM at theEuropean Union level, ICCVAM in the US and ZEBET (The German Centre for Documentation andValidation of Alternatives to Animal Experiments) in Germany1.
20.
After a test has been validated and its performance (relevance and reliability) has been determined
to be acceptable for its proposed use, a recommendation may be made that it be adopted as an OECD Test
Guideline. Sponsors of test methods proposed for adoption as OECD Test Guidelines should provide
evidence that the test method has been adequately validated in accordance with internationally recognizedprinciples and criteria, such as those described by the Solna Workshop (1). These criteria apply for both
new and revised tests. Regulatory acceptance is dependent upon the outcome of the validation, withconsideration of the principles given above. In considering the regulatory acceptance of a test method, thefollowing criteria are also important (1).
1) Application of the method provides data that adequately predicts the end-point of interestin that it demonstrates either a linkage between (i) the new test and an existing test methodor (ii) the new test and effects in the target species.
2) The method generates data for risk assessment purposes that are at least as useful as, andpreferably better than, those obtained using existing methods. This will give a comparable
or better level of protection for human health or the environment.
3) There are adequate testing data for chemicals and products representative of the type ofchemicals administered by the regulatory programme or agency (e.g., pesticides,
cosmetics).
4) The test should be robust and transferable and allow for standardisation. If highly
specialised equipment, materials or expertise are required, efforts should be sought tofacilitate transferability. This is an important criterion to be considered at an early stage ofa validation study. [Note added by the Secretariat: According to current OECD policy, thetest should not require equipment or material from a unique source. This would prevent theacceptance of patented methods. The Solna Workshop did not discuss the issue ofpatented tests but referred the issue to higher policy levels at OECD].
5) The test is cost effective and likely to be used.
6) Justification (scientific, ethical, economic) should be provided for the new method withrespect to any existing methods available. In this respect due consideration should be given
to animal welfare consideration including the 3Rs.
1

There are other organisations which contribute greatly to the development and use of reduction, refinement andreplacement alternatives by providing funding to research activities and/or organising activities that promote theprinciple of the 3R, such as the Johns Hopkins University Centre for Alternatives to Animal Testing (CAAT), theFund for the Replacement of Animals in Medical Experiments (FRAME), the European Research Group for
Alternatives to Animal Testing (ERGATT), the National Centre for Alternatives (NCA) in the Netherlands and theSwedish Animal Welfare Agency (SAWA).

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21.
Although the process of test validation, as described in this document, is separated into discrete
phases the test method validation process is, in practice, a continuum between the various elements ofvalidation. Each phase of the process uses and builds on results of other phases of the work. Regardless ofwhether the approach to validation is prospective, retrospective or modular (11), or otherwise, the goal ofthe validation process is to ensure that each of the OECD validation principles has been adequately
addressed. Therefore, it is essential that documentation of adequate validation be provided as outlined anddescribed in Chapter VII.
22.
Definitions of specific words and terms used in this document are presented in Annex I, where
examples of the variability of terminology used by different validation agencies are also briefly covered.
For examples of different validation studies including in vivo, in vitro health and ecotoxicity studies, seeannex 2.
23.
This document addresses the important steps and aspects that should be considered prior to andduring the validation process. They include:(i) The definition of the test method and related issues (e.g., purpose, decision criteria, endpoints,
limitations);(ii) the design and conduct of prevalidation studies leading to the optimisation of the test method;(iii) the design and conduct of the formal inter-laboratory validation work, based on theoutcomes of the prevalidation studies and aiming at accumulation of data on the relevance andreliability of the test method, and;(iv) the overall data evaluation and subsequent validation study conclusion, keeping in mind therequirements of regulatory authorities, for submission of information relating to new or modified
test procedures.
It also discusses the need for and the extent of an independent evaluation, or peer review, of test methods
being validated. This document provides a synopsis of the current state of test method validation.

16

ENV/JM/MONO(2005)14
II. DEFINITION OF THE TEST METHOD
24.
In the context of this Guidance Document, and of the overall OECD Test Guidelines Programme, atest method is an experimental system that can be used to obtain a range of information from chemical
properties through the adverse effects of a substance. The term “test method” may be used interchangeably
with “assay” for ecotoxicity as well as for human health studies.
25.
The planning and conduct of a validation study encompass and are applied to a wide range of test
methods and their procedures, which may be applied either as a stand alone test or as components of a test
battery or tiered test system. These considerations may include chemical properties, (Q)SAR procedures
and models, in vitro tests, in vivo animal tests, ecotoxicity and ecological test methods, and available
human data and test methods. In the future, new technologies such as genomics, proteomics and othernovel techniques may be incorporated into test methods. These test methods also vary in having single andmultiple endpoints and having different types of endpoints (e.g., cytotoxicity, tissue and organ weights,
histopathology grades, and serum and clinical analyses). Validation studies may address new tests,
substitute tests, modifications of endpoints in existing tests, and enhancement of existing test methods,
regardless of whether tests are for chemical properties, human health or environmental effects. The amountof information required in these validation studies, the number of chemicals tested, when and to what
extent to use testing, and the number of laboratories participating may vary as long as such factors do not
adversely affect the outcome of the validation process. For these reasons, the planning and conduct of avalidation study for a test method are expected to be done on a case-by-case basis.
26.
The first step in developing a new or revised test method is the definition of the test method. The
definition includes the test method’s chemical, biochemical or biological basis. This includes a rationale
for the relevance of the results produced such as the endpoints to be measured and a rationale or decision
criteria for how the results are to be interpreted and used, e.g., by using a data interpretation procedure(DIP) or a prediction model (PM)(3). The definition should also include the use of the results in theassessment of human health or environmental effects, and, as appropriate, the test method’s position
relative to other data/information requirements and test methods, e.g., location in a testing battery or tiered
testing strategy.
27.
To elaborate on the above points, an example of the basis for a test can be described by themechanistic or type of effects it is designed to measure (e.g., inhibition of the differentiation of embryonicstem cell lines, induction of mutation by base-pair substitution) (12). Examples of the role or use of thetest may be described in terms of:•

The biological effect it is designed to predict (e.g., hypersensitivity potential in people based on amechanism-based biological response in the murine Local Lymph Node Assay (13)).

•

The biological effect it is designed to measure (e.g., carcinogenicity in animals, toxicity in a
specific species of fish, alterations in one or more component of the endocrine system to an extent
that causes adverse health effects in the rat).

•

The species of selection in biological tests is designed to represent e.g., a special ecological
function found in healthy environmental communities, a special taxonomic group, trophic level or
microhabitat.

Examples of a test method’s scientific and regulatory needs include identifying whether a chemical might
have an effect on the liver, or an effect on the number of eggs produced by fish species. Examples of a test
method location in a tiered testing strategy might be as either in vitro or in vivo assays that might precede along term study of reproductive effects. A test method’s applicability domain refers for example tochemical classes, mechanisms of action and to the range of responses for which the test can be usedreliably. The applicability domain may also specify known limitations of the test method such as
17

ENV/JM/MONO(2005)14
restrictions on the classes of substances that can be accurately identified or measured by the test.
Furthermore, the test method may be suitable for identifying active substances, but not inactive substances,or vice versa.
28.
The following are more detailed descriptions of five categories of test methods and testing
strategies. In a strict sense, only the first three are categories of test methods and they may all be
replacement test methods and/or part of a test battery.
•A screening test method is a rapid, usually simple test performed for the purposes of prioritizing orgrouping substances in general categories of potential modes of action (e.g., in vitro binding to theoestrogen receptor). The results from screening tests are generally used for preliminary decision
making and to set priorities for additional and more complex tests. Although the results fromscreening tests, alone, may not be sufficient for risk assessment purposes, there may becircumstances where such results may be combined with other test results in a tiered testing
approach to provide in the hazard/risk assessments. As for all test methods, the limitations of thetest also need to be taken into account, for example, certain screening tests may only be able toidentify active substances, but not inactive substances. Other screening assays cannot distinguishagonist from antagonist activities (e.g., in vitro oestrogen receptor binding assays).

•

A definitive test method generates sufficient data to characterise the specific hazard of thesubstance without the option to always require further testing (e.g., two-generation reproductive
toxicity study). Ideally, it is a test method which provides sufficient stand-alone information ondose response and adverse effects to permit risk assessment and risk management organisations tomake decisions to reduce or minimize hazard and risk. However, in practice, data from other test
methods and experiments, if available, are typically included in risk assessment and management,
and the definitive test results do not stand alone.

•

An adjunct test method provides data that add to the data set or help interpret the results of othertest methods to assist the assessment process (e.g., toxicokinetics of an animal carcinogen tointerpret the carcinogenicity findings in different species).

•

A replacement test method is designed to replace an existing test method, whether it is a screening
or definite test. For a test method to be considered as a replacement, it should offer advantages
over the accepted test method. Such advantages may be a reduction in animal numbers or
suffering (i.e., consideration of the principle of the 3Rs), equal or more accurate prediction ofhuman health and environmental hazards, increased sensitivity or reliability over an existing test
method, increased information useful for support of decisions, decreased cost, or enhanced ease ofuse. The validation exercises should be sufficiently rigorous to demonstrate unambiguously thatthe new or revised test procedure meets or exceeds the performance of the test method to be
replaced.

•

A test battery comprises a number of test methods that are generally performed at the same time orin close sequence (e.g., genotoxicity test battery or aquatic base set tests that include data for algae,
daphnia, and fish species). Each test method within the battery is designed to complement theother test methods such as measure a different endpoint or mechanism of genotoxicity or adifferent component of ecological systems. Component test methods of test batteries are treated as
individual test methods for validation purposes and it is necessary to demonstrate that the
combination of test methods produces reliable and relevant results and is more effective than theindividual tests. In general, substitution of any component of the battery should improve its
performance.

18

ENV/JM/MONO(2005)14
29.
Current test methods may be periodically revised for reasons of enhancement and improvement toproduce an enhanced test method. Examples of enhancement could involve the addition of new endpoints
in order to increase the sensitivity of the test method or introduce augmentations of existing endpoints.
New methods or endpoints could also be designed, for example, to reduce the number of animals or toallow the assay to be performed in less time. As for a new test method, validation of new endpoints that
address new mechanisms and toxicities is needed so that the results can be interpreted with scientific andregulatory confidence. With existing complex test methods, the addition of new measurements or
endpoints that increase the complexity, both in logistics and interpretation, require appropriate
consideration during validation.

19

ENV/JM/MONO(2005)14
III. APPROACHES TO VALIDATION30.
To establish the scientific validity of a test method for a particular purpose, it is necessary to obtain
information that fulfils the criteria and scientific principles for test method validation, and to assess theextent to which the principles have been fulfilled. The information can be obtained by consulting existingsources (retrospective assessment of validation status), by generating new experimental data (prospective
validation studies), or by adopting a combination of prospective and retrospective validation approaches.
31.
The principles for validation apply to all test methods. Scientific rigour is always required,regardless of the scope of the validation, the type of test method, or whether the method is new, revised or
historical. Nevertheless, the level of necessary assurance that is appropriate for a specific purpose varies
and needs to be identified on a case-by-case basis.
32.
The amount and kind of information needed and the criteria applied to a new test method will
depend on a number of factors. These include:•

The regulatory and scientific rationale for the use of the test method;•

the type of test method being evaluated (e.g., existing test, new test);•

the proposed uses of the test method (e.g., mechanistic adjunct, screening or definitive test, total orpartial replacement of an animal or non-animal test method);•the proposed applicability domain of the test method (e.g., restricted chemical classes, multiple
chemical classes);•

the relationship of the test species to the species of concern;•

the mechanistic basis of the test and its relationship to the effect(s) of concern; and,•the history of use of the test method, if any, within the scientific and regulatory communities.

33.
For cases in which a new test method addresses an endpoint that has not been previously
considered or for which there are no or inadequate established reference data, a rigorous assessment of thepredictive capacity may not be possible, however, the relevance should be assessed using all available
information. Such tests that have shown to be reliable, but whose relevance has only been partiallyassessed by comparison with related reference data (e.g., TG 414 (14) and TG 424 (15)), should preferably
have their validation status periodically reviewed, with full consideration of all relevant available data. In
addition, in cases where it is contemplated enhancing an assay by adding new endpoints onto an existing
complicated study design some degree of validation is needed to demonstrate such additions arelogistically feasible, would not impair existing procedures and measurements and are not redundant.
Prospective Validation Studies
34.
A prospective approach to validation studies should be carried out when some or all of theinformation considered necessary to adequately assess the validity of a test method is not available, so thatnew experimental work needs to be performed. The data generated are used to determine the performance
characteristics (i.e., relevance, intra-, and inter-laboratory reproducibility), advantages, and limitations of
the test method. Following completion of a prospective validation study, an evaluation of all the available
information and data would then be conducted in order to assess the validity of the test method for a
specific proposed use.
35.
Before resources are invested into a prospective validation study (16) certain types of information
should ideally be available, including:20

ENV/JM/MONO(2005)14•

A clear statement of the scientific rationale and regulatory purpose of the test method.

•

An explanation of the need for the test method. The most obvious reason for the development of a
new test method is to address an area of toxicological concern for which tests do not yet exist.
Another reason is that the new test method or a new test species will have an advantage(s) overexisting test methods, such as its speed or ease of performance, reduced cost, reduction in numbersof animals utilised, refinement (i.e., reduced stress and pain in animals), increased sensitivity andaccuracy, improved identification of specific classes of substances, or its use as an in vitro
screening test or replacement for an in vivo test.

•

Clear and comprehensive standardized test method protocols, preferably in compliance with GLP
Principles, together with standard operating procedures (SOP), as appropriate. This should include
a description of the test system, exposure conditions, dose selection procedures, endpoint(s)assessed, measurements taken, specialized equipment or supplies that may be needed, measures ofvariability, the way in which the results are calculated and expressed, and the use of positive andnegative controls and other performance checks.

•

A description of the rationale and criteria to be used to analyse and interpret the test results. (e.g.,decision criteria, PM or DIP, where appropriate).

•

Background information supporting the expected or established usefulness and limitations of theselected test method. Included with this information should be a list of available publications andunpublished reports on the test method.

Retrospective Assessment of Validation Status
36.
A retrospective assessment of the validation status of a test method can be carried out by reviewing
all of the information and data available that supports or questions the validity of a test method, including
the results of any previous validation studies that have been conducted. In such a case, new experimental
work may not need to be performed, but additional data analysis and evaluation work may be necessary.
Whether or not additional validation is conducted should be decided on a case-by-case basis, andconsistent with the principles outlined in this document.
37.
For test methods in which there are published data available, the first effort should be to conduct athorough examination of the peer-reviewed scientific literature, and any other relevant and credible reports
and publications for information about the performance of the test method. This effort should beundertaken to determine whether sufficient information is available in the literature to substitute fully, orfor some components, in a prospective validation process. In some cases the published information islimited in description or in the presentation of test validation information, due to journal space limitations
and costs. Also, the validation data may be only available in the “grey literature”, e.g., in technical reports,
draft papers, and other miscellaneous unpublished documents that are difficult to obtain. Furthermore,these documents may be written in a language which would require translation. In all cases the principles
as articulated in Table 1 should be fulfilled for test methods without a full prospective validation study, butinstead, with a history of use and published data in the scientific literature. These principles are needed todetermine the performance characteristics, advantages, and limitations of any tests proposed for regulatory
adoption. Retrospective assessment of the validation status of these test methods will require obtaining thestudy protocols and independently analyzing raw data so that these critical features of validation can beadequately evaluated. For such a case, it may be useful to form an independent expert panel to evaluate theassembled data according to the validation principles described above, and to assess the reliability andrelevance of the test, and its advantages and limitations, without undertaking additional laboratory testing.
There may be situations, however, when data gaps need to be filled as part of the process to achieveadequate validation for a specific proposed use. In these cases, attempts should be made to limit the extent

21

ENV/JM/MONO(2005)14
of new animal testing as far as possible. Retrospective validation assessments should also be subjected toindependent peer review (see chapter V; Independent evaluation of a validation study: peer review).
38.
When conducting a retrospective assessment of the validation status of a test method, it may benecessary to address some special issues. For example, it may be necessary to determine the extent thatexperimental data obtained with different variants of a test method protocol can be pooled for the purposesof a single analysis, or whether an assessment of test method validity can be made in the absence of raw
data.
“Modular” Approaches to Assessing Validation Status
39.
The decision as to whether a retrospective assessment of validation status is adequate or whether
prospective validation studies are necessary depends on whether or not adequate reliable information isavailable to substantiate the validity of a test method. In practise, it is often necessary to adopt a
combination of retrospective and prospective approaches in order to generate adequate information and
data.
40.
A general conceptual framework for documenting the studies necessary to assess the validation
status of a test method, called the “modular approach” to validation, has been proposed (11). In thisapproach, information needed to support the validity of a test method is organised into modules thatprovide the following information:(i) Test definition (including purpose, need and scientific basis);(ii) intra-laboratory repeatability and reproducibility;(iii) inter-laboratory transferability;(iv) inter-laboratory reproducibility;(v) predictive capacity (accuracy);(vi) applicability domain; and,(vii) performance standards.
According to this proposal each of these “modules” of information should be available in order to assess
the validation status of the test method for a specific proposed purpose, but they need not be completed in a
linear/sequential fashion.

22

ENV/JM/MONO(2005)14
TABLE 1. PRINCIPLES AND CRITERIA FOR TEST METHOD VALIDATION
a)

b)

c)

d)

e)

f)

g)

h)

The rationale for the test method should be available.
This should include a clear statement of the scientific basis, regulatory purpose andneed for the test.
The relationship between the test method's endpoint(s) and the (biological)
phenomenon of interest should be described.
This should include a reference to scientific relevance of the effect(s) measured by thetest method in terms of their mechanistic (biological) or empirical (correlative)
relationship to the specific type of effect/toxicity of interest. Although the relationshipmay be mechanistic or correlative, test methods with biological relevance to theeffect/toxicity being evaluated are preferred.
A detailed protocol for the test method should be available.
The protocol should be sufficiently detailed and should include, e.g., a description of
the materials needed, such as specific cell types or construct or animal species that
could be used for the test (if applicable), a description of what is measured and how it
is measured, a description of how data will be analysed, decision criteria forevaluation of data and what are the criteria for acceptable test performance.
The intra-, and inter-laboratory reproducibility of the test method should bedemonstrated.
Data should be available revealing the level of reproducibility and variability within
and among laboratories over time. The degree to which biological variability affects
the test method reproducibility should be addressed.
Demonstration of the test method's performance should be based on the testing ofreference chemicals representative of the types of substances for which the test
method will be used.
A sufficient number of the reference chemicals should have been tested under code to
exclude bias (see paragraphs on “Coding and Distribution of Test Samples”).
The performance of the test method should have been evaluated in relation to
relevant information from the species of concern, and existing relevant toxicity
testing data.
In the case of a substitute test method adequate data should be available to permit a
reliable analysis of the performance and comparability of the proposed substitute test
method with that of the test it is designed to replace.
Ideally, all data supporting the validity of a test method should have beenobtained in accordance with the principles of GLP.
Aspects of data collection not performed according to GLP should be clearly
identified and their potential impact on the validation status of the test method should
be indicated.
All data supporting the assessment of the validity of the test method should beavailable for expert review.
The detailed test method protocol should be readily available and in the publicdomain. The data supporting the validity of the test method should be organised andeasily accessible to allow for independent review(s), as appropriate. The test method
description should be sufficiently detailed to permit an independent laboratory tofollow the procedures and generate equivalent data. Benchmarks should be available
by which an independent laboratory can itself assess its proper adherence to theprotocol.
23

ENV/JM/MONO(2005)14
Performance Standards for Test Methods
41.
The purpose of performance standards is to communicate the basis by which new test methods,
both proprietary (i.e., copyrighted, trademarked, registered) and non-proprietary can be determined to havesufficient accuracy and reliability for specific testing purposes. These performance standards, based on
validated and accepted test methods, can be used to evaluate the accuracy and reliability of other analogous
test methods (colloquially referred to as “me-too” tests) that are based on similar scientific principles andmeasure or predict the same biological or toxic effect (17). However, documentation as outlined in Chapter
VII should be developed to adequately address to what extent the validation and acceptance criteria havebeen met. Performance standards such as those described below should be provided, as appropriate, in Test
Guidelines issued for new test methods.
The three elements of performance standards are:Essential test method components: These consist of essential structural, functional, and proceduralelements of a validated test method that should be included in the protocol of a proposed,
mechanistically and functionally similar test method.
These components include uniquecharacteristics of the test method, critical procedural details, and quality control measures.
Adherence to essential test method components will help to assure that a proposed test method isbased on the same concepts as the corresponding validated test method.
Minimum list of reference chemicals: These are used to assess the accuracy and reliability of a
proposed, mechanistically and functionally similar test method.
These chemicals are a
representative subset of those used to demonstrate the reliability and the accuracy of the validated
test method. To the extent possible, these reference chemicals should:- Be representative of the range of responses and effects that the validated test method is capable ofmeasuring or predicting- Have produced consistent results in the validated test method and in the reference test method
and/or the species of interest- Reflect the accuracy of the validated test method- Have well-defined chemical structures and purity- Be readily available (i.e., from commercial sources)- Not be associated with excessive hazard or prohibitive disposal costs
These reference chemicals are the minimum that should be used to evaluate the performance of a
proposed, mechanistically and functionally similar test method. If any of the recommended
chemicals are unavailable, other chemicals for which adequate reference data are available could besubstituted. To the extent possible, the substituted chemical(s) should be of the same chemical classand activity as the original chemical(s). If desired, additional chemicals representing other chemicalor product classes and for which adequate reference data are available can be used to morecomprehensively evaluate the proposed test method. However, these additional chemicals should
not include any that had been used to develop the proposed test method.
Accuracy and reliability values: These are the comparable performance requisites that should beachieved by the proposed test method when evaluated using the minimum list of reference chemicals.
Use of Performance Standards for Catch-up Validation
42.
Subsequent to the determination that a test method is valid for a specific proposed use,establishment of performance standards based on this test method may be used to assess the validity of24

ENV/JM/MONO(2005)14
other structurally and functionally similar test methods. The identification and use of standard reference
substances to evaluate these similar methods (16) can facilitate so-called “catch-up validation” (18)(19).
This expedited approach has been successfully applied to the validation of a human skin model for skincorrosivity (20). Regardless of the approach used to assess the validation status of a test method, all new orrevised test methods should undergo a formal peer review. Each test method will need to be considered on
its own merits and should meet the general principles for test method validation.
Use of Performance Standards for Modified Test Methods
43.
As a new test method is used, the number of results obtained with the test expands. These results
should ideally be periodically reviewed to determine if the usefulness of the test method has changed. It
may be appropriate from time to time to review and reassess the validation status of established test
methods for hazard/risk assessment. Such reviews could be "event-driven" (in response to new scientificfindings individually suggesting a need for reassessment) or periodic (to determine whether the collectedbody of new information amassed since the previous review warrants a reassessment).It is also important
to be aware of possible changes over time from the original properties or responsiveness of the test system,or modification of procedures that might affect the outcome of the test method. Monitoring of positive,
negative and benchmark controls can aid in determining the occurrence or effects of changes.
44.
It may be appropriate to evaluate proposed potential improvements to an approved test method.
Prior to adoption of such changes, there should be an evaluation to determine the effect of the proposedchanges on the test’s performance and the extent to which such changes affect the information available forthe other components of the validation process. Depending on the number and nature of the proposedchanges, the generated data and supporting documentation for those changes, they should either be
subjected to the same validation process as described for a new test, or, if appropriate, to a limitedassessment of reliability and relevance using established performance standards. The extent of validation
studies that would be appropriate should be determined on a case-by-case basis. Major changes should be
subjected to a new independent peer review. This should take into account that the test was previously
adopted, and that the specific changes and enhancements (or improvements) proposed are intended withthe goal of improving the test’s performance. In addition, consideration should be given to such factors as
numbers of laboratory animals used, feasibility of the proposed revisions, cost and time effectiveness, andlaboratory capacity.
Validation of Patented Methods
45.
For validation purposes, patented or proprietary methods should be treated like other methods andthey should be scientifically valid for their proposed specific use. The OECD currently will not develop
Test Guidelines that require the use of a unique instrument or process owned by a patent. One reason forthis is that the method should be readily available to all potential users: another is to avoid market
monopoly of an OECD test method by a private company. One option to make it possible to adopt apatented test as an OECD Test Guideline would be to include a detailed generic description of the method
and provide proper reference to the validated, patented version of the method, together with a set of
performance standards (17). Any other version of the method, whether patented or not, should meet these
performance standards.
Validation of Test Batteries / Testing Strategies
46.
A test battery, base set or tiered testing approach is a series of tests usually performed at the sametime or in close sequence. Each test in the battery is selected so as to complement the other tests, e.g., toidentify a different component of a multi-factorial effect, or to confirm another test. A battery of tests may
also be arranged in a hierarchical (tiered) approach to testing. In a tiered approach, the tests are used25

ENV/JM/MONO(2005)14
sequentially; the tests selected in each succeeding level are determined by the results in the previous levelof testing. In these cases, a decision is made at each tier on whether there is sufficient information to stop
testing or whether additional testing is needed, at present this is a common approach for e.g., ecotoxicity
testing.
47.
Comprehensive guidance on the validation of test batteries and testing strategies has not beendeveloped. However, a few general considerations are given here.
48.
Individual tests within a battery of tests (or testing strategy) should be validated using thevalidation principles described in this document, taking into consideration their restricted roles in the test
battery/testing strategy. Justification for the acceptance of a test battery should be primarily on the basis of
its overall performance for its intended purpose. When tests are used in a tiered approach, the overall
results will normally depend on the strength of the individual tests in the tier unless certain tests in the tierare used in a confirmatory manner or have a “safety net” function.
49.
The performance of a tiered testing strategy, i.e., its predictive capacity and ability to replace or
reduce or refine the use of animals, may be evaluated by simulating possible outcomes of the strategy
using existing data, as exemplified by assessments of tiered testing strategies for skin corrosion(21)(22)(23) and for eye irritation (24)(25).
Validation of (Quantitative) Structure-Activity Relationships – (Q)SARs
50.
Structure-activity relationships and quantitative structure-activity relationships, collectively
referred to as (quantitative) structure-activity relationships, (Q)SARs, are theoretical models that relatechemical structure to physicochemical properties, environmental fate parameters, or biological activity.
Such models may provide useful predictions of such endpoints, and therefore reduce the need to obtain
experimental data for the purposes of hazard assessment.
51.
The specific issues associated with validation of (Q)SARs is addressed in a separate GuidanceDocument on (Q)SAR Validation that is being developed by the OECD, taking into account recent
publications in the (Q)SAR field (26)(27)(28)(29)(30)(31)(32).

26

ENV/JM/MONO(2005)14
IV. DESIGN AND CONDUCT OF VALIDATION STUDIES
General Considerations
52.
The purpose of the validation process is to determine the performance characteristics, usefulness,and limitations of a test method that is under consideration for use in a regulatory context, and to determine
the extent that results from the test can be used for hazard identification, and to support risk assessments orother health and safety decisions. Validation is distinct from test method development.
53.
The principles and criteria for validation are applicable to all test methods (see paragraph 17).
Scientific rigour is always required, regardless of the scope of the validation, the type of test, or whether
the method is new or revised. However, the level of assurance that is appropriate for a specific purpose andtype of test varies and should be assessed on a case-by-case basis. Some general guidance may be given,
particularly for certain types of studies.
54.
The key factors and workflow in the validation process are shown in Figures 1 and 2. Regulatory
authorities may have specific requirements for the analysis and summarisation of results of validation
studies prior to the submission of such studies.
55.
In certain situations an existing test that has been used as a research tool or in product development
will be proposed for use in a regulatory context. Although the test may have been widely used in the past,
there may not have been a systematic accumulation of data using a standardized protocol to allow a formal
evaluation of its reliability and relevance. The validation of such a test may proceed by two different
pathways, depending on the available database;(i) The review of all available data so as to perform a retrospective analysis of the reliability andrelevance of the test; or, if sufficient and appropriate validation data are not available,(ii) by undertaking additional prospective validation studies, in order to develop a data set of
relevant chemicals, to be considered together with previously existing data.
56.
In some cases a combination of both approaches might be appropriate in order to provide therelevant information necessary to assess the validity of a test.
57.
Regulatory acceptance is dependent on the outcome of scientific validation,. The acceptanceprocess will be greatly facilitated by the early involvement of regulatory authorities in the planning anddesign of the validation study so that it will address areas of interest and concern to the respective
regulators.
58.
Before subjecting a test method to a formal inter-laboratory validation study, standardization andan initial assessment of the proposed test method is recommended. This initial phase is often referred to as“prevalidation” and is performed for several reasons (33). Three of the most important are:(a) Refinement, optimisation and standardisation of the test protocol and standard operatingprocedures (SOPs), as appropriate, so that they can be readily used by other laboratories;(b) development of preliminary data on reliability and relevance of the test method; and,(c) avoiding the unnecessary expenditure of resources on multi-laboratory studies of methods ofdoubtful performance (Figure 1).

27

ENV/JM/MONO(2005)14
FIGURE 1. FROM TEST DEVELOPMENT TO VALIDATION: ENTRY POINTS FOR TESTMETHOD OPTIMISATIONTEST DEVELOPMENT
Initial Steps of the
Validation Process− Optimisation− Transferability− Reproducibility− Relevance

No

o

1

PREVALIDATION
Point 1Each element of a test (for example chemical exposure
time, histopathology, clinical chemistry, etc.,) needs tobe carefully explored and evaluated to determine theoptimum conditions/details.

2

Point 2At least one laboratory independent from the test
method developing laboratory will conduct the assay
for an initial assessment and review of its interlaboratory transferability and reproducibility. In casethe test method fails to provide sufficient
reproducibility, depending on the degree of failure, it
may or may not be considered for further optimisation(33, 34).

3

Test Method
performanceacceptable?

Point 3
Test methods may fail to show acceptable performancein terms of relevance rather than a lack of
reproducibility. This could be the result of a weaknessof the test method protocol. If further test optimisation
does not solve the performance issue, redesign of partsof the test method may be in order or a rejection of the
proposed test method should be considered.

Yes

Main part of the
Validation Process

4

Fully assess reliability +relevance

INTER-LABORATORY VALIDATION
Point 4
In certain cases where the result of the prevalidation
was not sufficient to justify further validation, but wasnevertheless promising, a test method could, afterrefinement, proceed to the main part of the validation
study, provided this phase is properly designed to deal
with the imperfections. In this case it is recommended
to perform the main validation study in more than one
phase.

One or more Phases including−Inter-laboratory
testing−
Blind testing−
Testing of positive
and negative control
substances

28

ENV/JM/MONO(2005)14
FIGURE 2. KEY FACTORS FOR VALIDATION AND REGULATORY ACCEPTANCE OF NEW
AND REVISED TOXICOLOGY TEST METHODS

Test Method Definition (Identify the Needfor Specific Data)•••

Essential Aspects Include:••

select/develop test
provide detailed description including
variables, as appropriate
collect background information
supporting the test selection•••

Initial Assessment of the Relevance andReliability of the Test Method Protocol(Prevalidation)
•
•
•

•

intra-laboratory testing
initial interlaboratory testing
test refinement/optimisation,
minimisation of protocol variables

Broad Assessmentof theRelevance
VALIDATION
Broad
Assessmentof andthe
Reliability
the Test Protocol
Relevanceand ofReliabilityof the Test
Protocol•inter-laboratory follow-up testing•
accumulationof follow-up
data on relevance,inter-laboratory
testing• reliability
accumulation of data on relevance,•
finalisation
reliability of appropriate test protocol•
finalisation of appropriate test protocol

•
•
•
•

•
•

Overall Evaluation and Conclusion•

independent peer review
conclusions of the validation study
recommendations
publication

•
•
•
•
•
•

Regulatory authority / Review andConclusion•

Recommendations for or against
proposed use

29•

identifying the basis of the test
defining the scientific purpose
and relevancedefining the endpoints andmeasurement
defining the test limitations
defining the rationale and decision
criteria in the interpretation of thetest results
defining the role of the test
designing the validation work:–
management structure–
GLP procedures–
selection of controls,
positive chemicals,
reference data–
blind testing–data collection andrecord keeping
procedures
identification of laboratories
participating in the work
optimisation of test method
protocols and development of
SOP’s
distribution of test
substances/chemical repository
perform testing
analyse dataassess reliability
assess relevancepeer review
defining the role of the test in
testing strategies
regulatory acceptance and use

ENV/JM/MONO(2005)1459.
Toward these ends the prevalidation process is also used to establish appropriate acceptable
response ranges for positive and negative control substances to develop the data recording and evaluation
criteria, to confirm or refine the preliminary decision criteria, to obtain a preliminary assessment of thetransferability, reproducibility, and reliability of the test, and to identify limitations of the test. Attention
should be paid to the latter point, because experience has shown that tests performing well in thedeveloping laboratory, or when used with only a limited range of chemical substances, may not perform as
well in a validation study as a result of limitations of the test that had not been determined and defined.
Other reasons for performing a prevalidation could include an assessment of a number of protocol
variations in order to select the most effective protocol(s) for the formal inter-laboratory validation study,
or to clarify aspects of the test protocol that may not be clear from the available information.
60.
After the initial evaluation has been completed, and prior to the planning and initiation of theformal inter-laboratory validation study, the results obtained should be fully evaluated, including anassessment of whether the conclusions of the study are supported by the data, and whether the proposed
protocol is adequate and appropriate. Based on this review, a decision would be made to:•Proceed to a formal inter-laboratory validation study using the selected test method protocol, SOPs
(as appropriate), and data evaluation or decision criteria as appropriate to maximize reproducibility
and accuracy.

•

Recommend further research/development to improve/modify the test method.

•

Proceed to an independent assessment of the test method, with the aim of having it accepted into
regulatory practice in the absence of a formal inter-laboratory validation study. This option would
be followed under circumstances where the results from the prevalidation study, pre-existing data,
and, if applicable, understanding of the test mechanism could support a recommendation that nofurther validation work is necessary.

•

Abandon the test method as not worthy of further validation.

61.
After prevalidation is successfully completed the formal inter-laboratory validation process may beinitiated (footnote; Ring-tests may be conducted in (a) prevalidation to develop the technical aspects of atest method or in (b) validation to evaluate the performance characteristics of a preferred method. The
results of the prevalidation will be used in the design of the formal inter-laboratory validation study. A wellplanned and designed prevalidation could lead to reduced cost and effort during the latter multi-laboratory
phase of the validation study.
62.
Following conclusion of the validation process, there could be a National or regional regulatory
acceptance process. For both sets of processes, there are principles and criteria that each test must meet
(Figure 2). Table 1 lists the principles and criteria for a valid test method. These principles and criteria
were adopted from a range of sources (1)(5)(6)(8)(16)(33)(35)(36). They provide a general description of
the information needed at various points in the test development and validation process. At each stage ofthe process, from definition of the test method through the various stages of validation to preparation of the
final report, the information provided should be sufficient to support a decision to proceed to the next stage.
63.
One conceptual approach proposed for managing and tracking the data from validation of a test
method is to collect the data and information in seven distinct “modules” as previously described inparagraph 43 (11). Irrespective of the approach used, a test method submitted for review of its validation
status should be accompanied by documentation outlined in Chapter VII (New Test Submissions:Supporting Documentation).

30

ENV/JM/MONO(2005)14
Management of Validation Studies
64.
Validation studies are normally conducted under the auspices of a sponsor, such as international
bodies, government entities and validation organisations for alternative methods (e.g., ECVAM, ICCVAM,ZEBET), national organisations, other independent organisations, or commercial sponsors. The sponsor ofa study will normally appoint a validation manager with well-defined roles and responsibilities, to coordinate the study (Figure 3). In the case of validation studies performed under the auspices of the OECDthe sponsor may be an OECD Expert Group, Task Force, Working Group, or Working Party whose
members are nominated by the governments of the respective Member countries. Member countries or
stakeholders, such as national and international organisations, industry associations, or non-governmentalorganisations may also represent the sponsor.
65.
The validation manager or management team should ensure that the purpose and objectives of thestudy are clearly defined, a project plan is developed, and a process for careful oversight of the validation
study is in place. The validation management should have collective expertise with the test, in theunderlying science and the scientific design, management and evaluation of a validation study.
66.
When conducting a validation study of a test method, ordinarily the sponsor would be expected tohave safeguards against conflict of interest in place to which the validation study should adhere. If it does
not, or they do not cover all aspects of conflict, individuals overseeing the study should not have personalor financial conflicts of interest with the test or the outcome of the validation study. Individuals responsible
for validation studies should have expertise with the test, in the underlying science and the scientific design,
management and evaluation of a validation study.
67.
The individual or team responsible for management of the validation should oversee the validationand serve as the central contact point for co-ordination of the testing by participating laboratories and forreceipt of data from those laboratories.
68.
The validation manager or management team may delegate essential tasks, such as the statisticalanalysis, the selection of test chemicals and selection of participating laboratories, including decisions
regarding the extent of GLP compliance to be used, to task groups or individuals.
69.
The validation manager should review and ensure the integrity of all information concerning thetest. Figure 3 demonstrates the role of the validation manager/management team in the validation process
and it summarizes essential tasks and interactions for managing validation studies. Schemes describing
various approaches for the organisation and management structure of validation studies have been
described elsewhere (5)(35)(37)(38)(39)(40)(41). Sponsors should ensure that all information and data
generated during validation of the test method are archived and are available for independent review.
Statistical Expertise70.
Sufficient statistical expertise should be available to ensure appropriate design of the validation
studies and evaluation of resulting data. A statistical advisor (biostatistician) should be a member of, or aconsultant to, the validation manager/management team and be involved in all phases of the validation of
new and revised tests. The statistical advisor should be familiar with the biological basis and the practicallimitations of the proposed test and of the reference test or endpoint in the ultimate species of interest.
This knowledge will aid in the selection of appropriate statistical procedures, and the development ofappropriate decision criteria, and can help with communicating the results of the study.
71.
The biostatistician should be involved in the design of all phases of validation studies and in theestablishment of appropriate procedures for record keeping, data collection, and data submission. The
31

ENV/JM/MONO(2005)14
statistical methods to be used should be defined before the start of the studies and be used to support the
proposed study design. However, because the specifics of the data can not always be anticipated, it may benecessary to revise the statistical procedures or identify or develop new procedures or models after receiptof the test data. The statistical evaluation should convey the reproducibility of the study by assessing
variability within and among laboratories. Acceptance criteria should be given at the beginning of thestudy as to the degree of variability and agreement judged acceptable for a valid test.
72.
At various stages in the validation process, statistical assessments should be made as to how wellresults from the new or revised test agree with the reference data using the decision criteria. In addition,
the test conditions should be evaluated statistically to provide recommendations for improving theefficiency of the protocol. Such an analysis can also serve to optimise and reduce animal use, for example,
by showing the power of the test as a function of the number of animals per group in relation to thereproducibility/variability and response of the endpoint that is being measured.
Conduct of the Prevalidation
73.
The prevalidation could be performed by one or more laboratories experienced in performing tests
similar to the new or revised test. The exact design of the study, the number of participating laboratories,
the laboratory qualifications required, the number of substances tested, and the number of animals to be
used, where applicable, may depend on the specific test, its proposed use and/or biometrical considerations.
74.
The prevalidation usually includes participation by one or more laboratories and/or experts inaddition to those who were responsible for developing the test. The extent of activity at this phase willvary greatly depending on the specific test and the amount of information already available on its
performance, and it may involve a single stage or multiple stages of experimentation, each building on theprevious phase of work.
75.
Additional activities at the prevalidation stage may include an assessment of the transferability ofthe test protocol to laboratories inexperienced in the test or the necessary techniques and to resolve
questions or inconsistencies arising in this phase. This process may lead to the development of an
optimised test protocol and accompanying SOPs, as appropriate, and may provide for development ofadditional data to be used for refining the evaluation and decision criteria for the test.
76.
For certain tests, training of the personnel in the participating laboratories and co-ordination ofscoring criteria and nomenclature would be an essential part of the study.

32

ENV/JM/MONO(2005)14
FIGURE 3. ROLE AND INTERACTIONS OF THE VALIDATION MANAGER/MANAGEMENT
TEAM

Sponsor of the Validation Study

Validation Manager/Management Team
•
•
•
•
•
•
•

Overall co-ordination/management
Approval of study design, protocols, time schedules, participating
laboratories, etc.
Test substances approval
Establishment of sub-groups (e.g., a task group for chemical selection
management)
Data evaluation/interpretation
Information exchange
Approval of all reports from the study

Task Groups

Lead Laboratory••••

As appropriate, e.g.;• Test substance selection• Test substance control(Repository)• Data analysis/evaluation• Performance summary

Training/Instructions
Co-ordination of SOP
Data collection from laboratories
Troubleshooting

Participating Laboratories
•
•
•

33

Data collection
Study conduct
Data evaluation

ENV/JM/MONO(2005)14

Project Plan
77.
The objectives of the validation study should be specifically defined and presented such that they
represent a clear, unambiguous and achievable goal. The validation management established for the
project is responsible for the definition of the purpose and objectives of the validation. A project planshould be produced so that all involved parties will have a clear understanding of the validation work to be
performed. The project plan will form the basis of an agreement among the sponsors, the validation
management group, and the lead and participating laboratories. The objectives and specific details of the
project and the protocol(s) to be used should be available to all study participants, who should agree, in
advance, to carefully follow the project plan and test protocol.
78.
In addition to the principles and criteria for a valid test method presented earlier in the text, thefollowing specific indications of readiness should be available before a test is accepted for inclusion in aformal inter-laboratory validation study:•

Definition of the test including its position in a testing program, its advantages and limitations, andthe classes and types of test substances that can and cannot be tested.

•

Existence of data evaluation or decision criteria based on the results of the prevalidation, which isappropriate for the data generated by the test and the effect to be predicted, modelled or measured(e.g., PM or DIP, where appropriate).

•

The specific, detailed protocol or protocols proposed for the inter-laboratory phase of thevalidation study, including SOPs (as appropriate), the measurements to be performed and thespecific types of data to be provided by the test.

•

Evidence of intra-laboratory reproducibility of the test, and an initial assessment (prevalidation) of
the inter-laboratory transferability.

79.
The project plan should contain essential information for the participating laboratories and describe
their duties and responsibilities; it should be updated, as needed, as the planning progresses. Depending on
the anticipated size and complexity of the study, essential tasks such as the selection of test chemicals,
development of a reference data base, selection of a lead laboratory, development of minimum criteria for,and selection of, the other participating laboratories, and selection of data analysis procedures and theperformances of statistical analyses, can be delegated to sub-groups or individual members reporting to thevalidation manager (figure 3).
Participating Laboratories
80.
Laboratories participating in the validation study should meet minimum standards in terms of theavailability of competent staff, facilities, safety, animal welfare (where applicable) and quality assurance
procedures. Ideally, the participating laboratories should have demonstrated competence with the generaltest method or similar methods and should provide, as a minimum, information on the adequacy of theirdata documentation procedures, such as their compliance with Good Laboratory Practices (GLP). It is also
important to consider that the choice of an inexperienced laboratory may have an impact upon the resultsof the validation. Ideally, validation studies should be performed, and data recorded and maintained, in
accordance with GLP (42)(43)(44)(45)(46)(47). As a minimum, they should always be conducted inaccordance with GLP principles, such as, but not necessarily limited to, use of SOPs, adequate data
recording and record keeping. The use of standardised software programs for data collection may be usefulin this regard.

34

ENV/JM/MONO(2005)14
81.
It is important that all laboratories participating in a validation study have a clear understanding of
their specific obligations and responsibilities. These include the requirement for strict adherence to test
method protocols, the prescribed timetable for submission of data, clarification of ownership of results, andpublication procedures (e.g., clearance, publish jointly or independently). It is a distinct advantage to havesuch issues clearly addressed in formal agreements or contracts, prior to the start of the study. In addition,
an international study may involve many languages and legal systems, as well as different financial
practices and contractual systems, all of which can complicate validation management if they are notformally agreed to in advance.
82.
The minimum and maximum number of laboratories needed for a comprehensive assessment of
the validity of the test method will depend on the type of test, the questions being addressed, the overall
amount of testing required of each laboratory, the cost of testing, and animal consumption. In many cases,
three or four laboratories per test method may be an adequate number for an assessment of the interlaboratory reproducibility. However, the assessment of the predictive capacity (performance of the test
method) may be conducted in fewer laboratories if the data are adequate. Having too many participating
laboratories may increase costs, logistical complexity, and animal numbers without necessarily improving
the scientific quality of the outcome. Alternatively, having too few participating laboratories may result ininsufficient data for an accurate assessment of the inter-laboratory variability. The decision as to thepreferred number of laboratories for each validation study should be based on bio-statistical considerations
and the issues enumerated in this paragraph adequate in a statistical sense.
83.
A lead laboratory should be appointed, and this laboratory should have demonstrated expertise andexperience in the discipline to which the test method belongs. It should also have proven experience in
conducting the type of test method under evaluation. Validation managers should oversee the development
or refinement of the initial protocol and ensure that appropriate training is provided, as necessary, topersonnel from the participating laboratories, so that all laboratories perform the test and evaluate the dataaccording to the same procedures and criteria (figure 3). The study director in each laboratory should
ensure clear communication with validation management.
Reference Data
84.
Relevance needs to be determined for all types of assays. For replacement tests, there should be anadequate reference data base generated in the original test method against which the new (or revised) test
method is compared. This reference data base should cover the expected range of responses (strong,
moderate, weak and negative) and the range of chemicals to which the assay will be applied (the domain of
applicability). A reference data base of this type should provide a measure of the performance of the test
method against a wide range of biological and chemical activities. For higher level in vivo assays, thenature of the endpoint, a direct measure of toxicity (e.g., histopathological change, organ weight change,etc.) would permit the option of a less extensive data set to assess relevance. Such data sets usually consistof the minimum number of chemicals necessary to cover the range of responses. Inter-laboratory
validation studies, when necessary, of such assays should be designed to generate the requisite data toestablish the relevance and reliability of the test method while taking into account practical limitations,such as animal use.
85.
The ultimate goal of toxicity testing is to protect human and ecological health and safety. For atest that is being designed to replace an existing test, all efforts should be undertaken to obtain reference
data from the existing test and, where feasible, the ultimate species of interest. While it is theoreticallypossible to directly test the species of interest in ecotoxicological tests, standard surrogate species are
generally used because they have been adapted to culturing in the laboratory and it is impossible to test
every fish or bird of interest, even if suitable number could be obtained from wild populations.

35ENV/JM/MONO(2005)14
86.
For assessing human health effects, data from humans are most relevant;. Human data may beavailable from several sources (50) including:•

Epidemiology•

Occupational exposure•

Accidents and cases of poisoning•

Clinical studies•

Ethically approved studies in human volunteers

The use of human data raises a number of issues that needs to be addressed, however, in practice it isnormally difficult to obtain high quality human data:•

The special challenges of retrospective data comparison;•

The lack of available human data on chemicals that do not show the effect of interest, so that adetermination of that test’s false positive rate cannot be made;•The ethical issue of human data development, including national ethical differences in how toapproach the use and acquisition of human data,•

The potential lack of accurate information on chemical exposures.

Consequently, the absence of sufficient human data may mean that animal data are the default reference.
For example, if the goal of a study is to replace an animal test that is being used as a surrogate for humaneffects, and human data are lacking, then data derived using that animal species may be the most relevant
available and should be used.
87.
Classifications of chemical activities in the reference species should be supported by detailed
information. The number of reference chemicals meeting these criteria may be insufficient. In such cases,
careful judgements including considering animal welfare should be made regarding the need ofdeveloping additional reference data.
88.
In ecotoxicology, the replacement test issue and process is currently handled somewhat differently
compared with other areas. Since there are so many different species of plants and animals occurring in theenvironment, and only a few from each group can ever be tested, much work has been expended in trying
to develop a minimum of tests for each group of organisms. Once these initial tests have been developed,
depending on the “importance” (in the ecological and economic sense) of a group, additional species in thesame group can be investigated for development as additional test species. Thus, for an important group oforganisms (e.g., fish) there is often a list of acceptable species that can be tested. It is up to the regulatory
authority to provide guidance on which species should be tested, and when in the hazard
identification/assessment process this testing should be conducted. Replacement tests should be based on
the same type of organism, e.g., a crustacean replacing an existing crustacean test, however, these direct
replacements seldom occur in ecotoxicology.
Selection of Test Chemicals and Chemical Management
89.
The validation management has the overall responsibility for organising the selection of chemicals
to be used. In general, a chemical selection sub-group may be established for that purpose (figure 3).
Because regulatory authorities are the future users of a new test method undergoing validation, it isadvisable to involve experts from these authorities in the selection of the test chemicals. Bio-statisticaladvice should be sought to determine the minimum number of test chemicals necessary for achieving theobjectives of the study. This number may need to be corrected if it does not allow a sufficient36ENV/JM/MONO(2005)14
representation of relevant chemical groups. This number might vary depending on whether the predictive
capacity (performance of the test method) and intra- and inter-laboratory reproducibility have to beassessed concomitantly or independently. Consideration could also be given to the establishment of achemical repository to purchase, code, and supply the test chemicals and relevant safety information to theparticipating laboratories.
90.
The chemicals should be selected based on the objectives of the study, and the type of test method
undergoing validation (49). Therefore, it is imperative that selection of the chemicals to be tested isconsistent with the defined objectives of the study. This means the chemicals need to be relevant to theadverse effect of concern, or mechanism of concern, and also be relevant for the species of concern.
Chemicals for which toxicological hazard assessment can be made based on measures other than biologicaltests (e.g., accepted physico-chemical properties or chemical structure characteristics) should not be
selected (or kept at minimum), for example, chemicals corrosive to skin because of their extreme pH
values. Chemicals should be chosen according to the availability of relevant and reliable reference data,
which ideally provide an unequivocal assessment of their activity in the reference test or in the organism(s)of concern. The performance of the test method being validated will be judged on the basis of its
responses to these chemicals, so the spectrum of chemicals and chemical classes that are ultimately
included in the study should be relevant to the chemicals for which the validated test will be used. In
making these selections, it is important to include a sufficient number of positive and negative chemicals
from the reference data. In all cases the criteria and reasoning for selection of test chemicals should betransparent and properly communicated, and included in published reports, if appropriate.
91.
The chemicals tested should, where possible, be of the highest available purity, or of known
composition. All laboratories in the validation study should use, to the extent possible, the same batch of atest chemical. Ideally, each test chemical should be as close as possible, in terms of chemical compositionand physico-chemical properties, to the material employed to generate the reference data. However, it
often will not be possible to use the same batch or exact formulation as was used to generate the reference
test method data. The chemicals should be readily available for testing (preferably from commercial
sources) and in sufficient quantity for use by all participating laboratories, including a back-up sample tocover unexpected losses and, to a limited extent, follow-up investigations that may become necessary.
They should be stable under the defined conditions of storage for at least the duration of the validation
study. Physico-chemical information regarding stability, solubility, volatility, etc., should be provided,
where known, as should storage, use, and disposal recommendations.
92.
The number of chemicals that should be used to characterise accuracy and reliability of a test
method will vary with the purpose and nature of the test method. For the assessment of inter-laboratory
reproducibility a subset of test substances used to assess accuracy might be appropriate, provided that thesubset adequately represents an appropriate range of responses and physical/chemical properties for which
the test method is proposed to be appropriate. In the validation of certain in vivo tests, such as
carcinogenicity tests and neurotoxicity tests, where the endpoints of interest are directly observed,
chemicals used in validation studies should cover the expected range of responses (such as strong,
moderate, weak and negative) and a rationale should be provided for the number and properties of thesubstances used. In addition, the experience gained during test development and prevalidation should betaken into account when selecting test chemicals, so as not to waste time and resources. The number, andtypes, of test chemicals will depend upon many factors, including;•

The type of test being validated;•

the number of participating laboratories;•

the number of toxicity hazard classes to be predicted;•

the range of toxic effects and potencies being investigated;37

ENV/JM/MONO(2005)14•

the diversity of chemical classes of interest and structural relationships among the chemicals;•

the range of physical states to be considered;•practical limitations pertaining to the complexity and duration of in vivo tests,•

the availability of relevant and reliable reference data for the chemicals; and•statistical considerations.

Coding and Distribution of Test Samples
93.
Chemicals to be tested should normally be coded in validation studies, unless precluded byscientific, technical or safety considerations. Testing laboratories should ensure that there is compliancewith the regulations for the transport or storage of dangerous chemicals in the relevant country. The
substances to be tested should be independently coded with a unique code, and packaged in a manner thatwill not reveal their identities (51). For safety considerations, and to aid in the selection of test article
diluents/vehicles, handling, and disposal procedures, sufficient information about the physico-chemical
properties of each test chemical should be made available to the participating laboratory and to thelaboratory’s safety officer. It is preferable to have the test substances coded prior to being sent to thelaboratories. A less desirable option is to have the designated safety officer of the testing laboratory orother individual not involved with the validation study apply the codes to the chemicals. In all cases, it isrecommended that Safety Data Sheets (SDS) for all chemicals to be studied be kept by the designatedsafety officer in each participating laboratory. Member countries may have specific regulations regarding
provision of SDS.
94.
The coded test substances should be transported in suitable containers, preferably from a central
repository, and should be labelled in accordance with relevant transport regulations (52). Laboratories
should be provided, where appropriate, with the information about each of the test chemicals, including:a) Visual appearance;b) physical state;c) weight or volume of the sample of test chemical dispatched;d) physico-chemical data, such as pH, volatility, and stability, solubility, chemical reactivity; ande) storage instructions.
95.
If specific chemicals require additional safeguards or control, the relevant information should be
included with the physico-chemical information sheet unless this would reveal the chemical’s identity, inwhich case an alternative means should be established to ensure safe use, handling and disposal.
Participating laboratories should be provided with a sealed package containing necessary information
about the health hazards of the test chemicals. This should include clear instructions for action in the caseof accidents, and information needed for decontamination or disposal of contaminated animals andlaboratory supplies, and of excess chemicals and working solutions. Advance notification of shipping,information regarding the storage requirements, and safety-related information about the specific chemicals,
should be provided.
96.
For validation studies that use coded hazardous chemicals, it may be advisable to treat all codedchemicals as if they have the same toxicity as the most hazardous chemical in the group. In such a case,health and safety information provided to the laboratories on this chemical should be considered to apply
to all the chemicals in the group. In certain situations, such an approach will not be possible (e.g., theemergency response procedures for one or more of the chemicals differ from those for the most hazardous
chemical on the basis of significant differences in water solubility). For those situations, one approach is38

ENV/JM/MONO(2005)14
to provide a sufficient number of sealed envelopes for each coded chemical separately, containing healthand safety data and marked with the code number for that chemical. The sealed envelopes should beavailable in all working areas where the chemicals are handled, to be available in case of an emergency.
However, laboratory staff should be instructed to open and read a given envelope only under such an
extreme situation. At the conclusion of the study, the participating laboratory should return all envelopes(sealed and open) and provide written explanations for every opened envelope. In some cases, a specifichealth and safety plan may need to be developed by the testing laboratory, in which case the health andsafety staff, but not the testing staff, would need to know the identity of the test articles. In all cases,
information provided to the participating laboratories should be in accordance with national and local legal
requirements.
Test Method Decision Criteria and Data Interpretation
97.
The outcome of prevalidation studies will determine if a test method is sufficiently developed, toproceed to an inter-laboratory validation study. A rationale and decision criteria for interpretation of test
results, classification of a chemical (e.g., positive, negative, equivocal) and a determination of toxicity
values and categories should be clearly defined in the standardized optimized protocol used for thevalidation study. It is noted that the decision criteria for the interpretation of test results, generally an
integral part of validation, should be transparent and unambiguous. Depending on the nature of data
generated by the test and also by the sort of test used, different approaches may be used to define therationale and decision criteria. In those cases where a toxic end point of interest is monitored or measured,
often in in vivo or ex vivo systems, clear decision criteria e.g., for positive, negative or equivocal outcomes,
should be defined (e.g., in a DIP).
98.
When the toxic end point of interest is not measured or monitored directly, an algorithm may be
used to convert the result obtained from using the test method into a measurement or prediction of the toxic
effect of interest. An algorithm is often available for tests, such as in vitro tests, where the results expressed
as a value, or a range of values, are related to a toxic effect of interest. However, they are rarely used for
ecotoxicity in vivo studies. During validation of the 3T3 NRU Phototoxicity test (OECD Test Guideline432) it was shown that properly developed probability-based PM’s can discriminate between treated anduntreated controls (53)(54)(55)(56).
Monitoring of Participating Laboratory Performance99.
In a study that uses a large number of test substances, it may be most effective to proceed in a stepwise fashion. Using this approach, only a subset of the substances is tested at first, and a compliance checkis performed on each laboratory, which includes a review of the complete data sets. After this review iscompleted, a decision is made as to whether each laboratory is performing satisfactorily. Laboratories thatare not able in this first step to perform the procedure according to the required protocol and SOPs (asappropriate), or maintain the appropriate record keeping procedures, should not be considered for further
participation. In this case, decisions must be made about whether the data from such laboratories are
sufficiently robust to use further in the validation process.
100.
At interim times during the course of the testing, validation management should determine whether
the laboratories are following the agreed test protocol and record keeping procedures. This can usually be
accomplished through compliance statements from the laboratories. If considered necessary, a compliancecheck may be performed, which could include a review of the laboratory’s records for a limited number oftest samples. Additionally, the quality of the laboratory's work (e.g., levels of contamination of samples,
performance errors, high reproducibility of data points) may be assessed. Deviations from the test protocol
or record keeping procedures, or evidence of poor laboratory practices, should be identified anddocumented. A decision should be made whether to correct the problems and continue the testing (by39ENV/JM/MONO(2005)14
retesting the inadequately-tested samples) or to eliminate the inadequate data from the validation study.
Alternatively, data that were not obtained using the required protocols could be identified but not used insubsequent analyses, and the reasons for their rejection documented. As a general principle, all data fromall participating laboratories will be used in the analyses and reports prepared by the lead laboratory or thevalidation management group.
Inter-laboratory Testing101.
Testing in this phase would not normally proceed until the validation manager is satisfied that thetest method protocols are clear and unequivocal, and that the tests can be adequately performed by allparticipating laboratories (for ring-tests, see paragraph 64). Moreover, validation management should
confirm that the procedures to be used for collecting, analysing, and reporting the data are adequate. A
definitive schedule should be established so that the test articles are supplied to, and received by, alllaboratories within a specific time, and that all laboratories complete their testing assignments within a
specified time. At the conclusion of the testing, or at agreed-upon intervals, the full data records oncompleted samples should be submitted to validation management or the data analyst using the appropriate
format, as described in the project plan.
102.
Laboratories that develop or validate test methods may not all be familiar with GLP or not be
organised to perform studies under strict GLP compliance. Components of GLP that should be followed in
all cases are that all protocols, experiment-related notes, and data entries should be complete, detailed,accurate, and annotated with the names of the individuals conducting the work, entering the data and thedates of the work and that all staff involved are adequately trained.
Data Collection
103.
If the validation study has a large number of participating laboratories, or if there is an anticipatedhigh volume of test data, separate task subgroups may be responsible for aspects of the study relating to theinter-laboratory co-ordination, training, and/or data collection, management and analysis. In certain cases
an independent contractor may be designated to receive, process, and analyse the data and may also serveas the lead laboratory, in accordance with the requirements on transparency(figure 3).
104.
The following factors should be addressed when planning a validation study and designing the datacollection and evaluation procedures.
•

Data to be collected, the data recording procedures, and the procedures for performing data quality
checks and analysing the data should be explicitly identified.

•

For each test method procedure the number of repeat experiments each laboratory will perform, ifany, and the number of independent replicate measures of the endpoint within an experiment, if
any, should be specified.

•A standard format for data entry to be used by all participating laboratories should be specified.
Care is needed to ensure that such forms and formats are not ambiguous; clear directions should begiven on how to report the data and the other test-related information.

•

The quality assurance and quality control procedures to which the original data obtained and any
values derived there from should be identified.

105.
Specific computer-based systems may be used to collect, analyse, and report the data, but the useof such a system should be agreed upon as part of the project at the study design stage. The study plan
approved by validation management should specify the data that is to be submitted and, on request, all40

ENV/JM/MONO(2005)14
individual data should be available. Unless otherwise specified in the study plan, all data should besubmitted. Where summary or transformed data are submitted, the original data should be available foranalysis.
Data Analysis
106.
The statistical analysis procedures used should be relevant to the types of data being analysed andthe questions being asked of the data. The study plan approved by validation management should specify
whether the participating laboratories will conduct analyses of their test data (according to the agreed-uponprocedures) or whether independent statistical analyses will be conducted. The latter approach is favoured.
The biostatistician(s) should be sufficiently knowledgeable about the test procedures and the design of thevalidation study to perform the appropriate analyses and to recognise anomalies in the data. If necessary,
provision should be made for validation management’s biostatistician(s) to directly contact individuallaboratories to clarify aspects of their data reports.
107.
In general, two types of information are required from a validation study, namely measures of test
reliability or reproducibility, and measures of the relevance of the test method. An assessment is made of
the qualitative and quantitative reproducibility of the test between the participating laboratories, including
measurements of the variability, within each, and between the different laboratories. Where appropriate, anassessment may be performed of the various parts of the protocol that may be most responsible for
affecting reproducibility and that contribute to the variability.
108.
The individual laboratory data are analyzed to determine the relevance of the test results. Where
applicable, this will include assessing the accuracy of the proposed test method for predicting the outcomeof the reference test method. Any adjustment of the decision criteria (e.g., PM) during the study should befully documented and scientifically justified. At the completion of a validation study, there may besituations where the data clearly indicate that the decision criteria need to be refined in order to increase thepredictive capacity for the intended use of the test method (57). The important consideration is that any
changes in the decision criteria need to be properly documented, and the effects and consequences of thechanges need to be clearly described. In terms of ecotoxicological test methods, decision criteria are often
used in a different manner from that used in human health testing. In ecotoxicology, decision criteria may
not be an integral part of the validation where the test results do not require interpretation.
109.
Several different statistical methods may be used for the analysis of data. The selection of the mostappropriate statistical approaches depends in part on the nature of the data and also on the design of thevalidation study. Qualitative or categorical data have often been described in terms of the Cooper statistics(58)(59), such as the sensitivity and specificity of the test results. There are many procedures that can beused to evaluate quantitative data. The specific procedure(s) used should be relevant to the types of data
being analysed, the comparisons being made with the reference or other data, and the purposes of the study.
110.
Following completion of the inter-laboratory phase of the validation, and compilation and analysisof the data, validation management should make a critical assessment of the outcome of the study. All datafrom each participating laboratory should be assessed and all factors should be taken into account todetermine whether the original goals of the study have been met. This assessment should review every
aspect of the validation study in a systematic manner.
Reporting111.
At the completion of the study all data and laboratory records should be archived. The goal should
be to have archived data and other laboratory records available, upon reasonable notice, in an easily
retrievable format for independent assessment.
41

ENV/JM/MONO(2005)14

112.
The results of the validation study and final conclusions should be discussed with all participantsof the study. Validation management should attempt to reach consensus on the results and conclusions of
the validation study before drafting the study report. The draft study report shall be circulated to the leadlaboratory and every participating laboratory for review and comment prior to finalisation. Validation
management should review all comments received and make revisions as the group deems appropriate.
113.
The final study report describing and discussing the validation study, the results obtained and therecommendations of validation management should be submitted to the sponsor. The final report should be
subjected to evaluation by an independent panel of peer reviewers (see chapter V; Independent evaluationof a validation study: peer review) prior to submission to regulatory authorities for regulatory acceptanceconsideration. The report of the independent peer review and the report of the validation study should bemade publicly available and, preferably, an article should be prepared for submission to the scientific
literature based on the report.
Record-keeping/Data Dissemination
114.
It is preferred, but not always strictly required, that validation studies are performed and reported
in accordance with the OECD Principles of Good Laboratory Practices (43). The records should contain
GLP compliance statements, and indicate if, and when, coded chemicals were used. Aspects of datacollection not performed according to GLP should be fully described, along with the potential impact of
not using GLP.
115.
The data supporting the test being validated, the detailed protocols under which the data were
produced, and all other supporting documentation, should be available for review, in accordance with therequirements described in the study plan approved by validation management. If mathematicallytransformed data or summary conclusions are provided, they should be linked to the original data.
116.
Preferably, there should be dissemination of summary information to interested parties on acontinuing basis throughout the validation study, as long as this would not compromise the study. Suchinformation could include:(i) A public announcement of the undertaking of the study, including its objectives, if consideredappropriate;(ii) reports of the progress and outcome of the study; and,(iii) publication of a report of the study in the peer-reviewed scientific literature.
Following completion of the validation study and peer review, the publication of a summary report of thestudy, and reports from the participating laboratories, should be encouraged. In addition, the study sponsor
should consider how to contribute to the transparency of the validation study. Some validation processes
make data available for review by interested parties by depositing such data in a repository open to the
public.

42

ENV/JM/MONO(2005)14
V. INDEPENDENT EVALUATION OF A VALIDATION STUDY (PEER REVIEW)
117.
Independent scientific evaluations (peer review) of test methods may be conducted to assess theextent that a proposed test method has addressed established validation and acceptance criteria for a
specific proposed use, and to provide expert advice on the proposed test method. This independent
assessment is normally conducted by a panel of qualified scientists that have not been involved in thedevelopment or validation of the test method, and who do not stand to benefit financially or professionallyas a result of the outcome of the review. Independent peer review provides a critical assessment of theusefulness and limitations of a test method for its proposed use, and aids regulatory authorities indetermining the acceptability of a test method. This process is an important step following the validation ofa new or revised toxicology test method. All aspects of all peer reviews should be fully transparent andallow for consideration of stakeholders comments.
Mechanisms for Peer Review
118.
There are several options for independent peer review of test methods in the development ofOECD Test Guidelines, including:(i) Peer review co-ordinated by an organisation established or mandated by law to evaluate test
methods;(ii) peer review co-ordinated by a government agency of a Member country; and,(iii) OECD co-ordinated peer review.
119.
Sponsors or other appropriate parties could contact organisations that specialize in test method
evaluations, e.g., ECVAM or ICCVAM, to arrange for an independent peer review. These organisations
provide a resource for assessing the validation status of test methods and a source of guidance for otherorganisations and can conduct and assess validation status. .
120.
It is important for the sponsor of the test method to carefully document the data available tosupport the validation review process. Principles and criteria described in table 1 should be met and thisinformation should be provided to the peer reviewers. Accordingly, a submission data package
documenting the validation status should include:•

Scientific and regulatory rationale for the proposed test method•

Rationale for essential test method protocol components•

A complete description of the substances used in the validation of the proposed method, and therationale for their selection•

In vivo or other appropriate reference data for test substances used to assess the test method’s
accuracy (where appropriate)•

All available data and results from the proposed test and reference methods•

Test method performance (accuracy)•

Test method reliability (repeatability/reproducibility)•

Statement assessing test method data quality•

Other pertinent scientific reports and reviews•

Assessment of test method refinement, reduction and replacement

43ENV/JM/MONO(2005)14•

Evaluation of usefulness and limitations of the test method•

References•

Supporting materials

121.
Practical guidance for organizing test method submissions that has been found to be helpful inassuring that peer review panels have sufficient information to evaluate the validation status of test
methods has been developed and used effectively by validation agencies such as ICCVAM (60). While
this should not be understood as a binding standard for other parties, it should be considered as one way toprovide adequate documentation for peer review (see also paragraph 145-146, Criteria for regulatory
acceptance).
122.
The party that submits a test method to the OECD Test Guideline Programme as the basis fordevelopment of a Test Guideline should indicate if peer review has already been conducted or propose apeer review approach to be used for its evaluation. Where peer review has been completed, a full andcomplete report of the independent panel should be provided, including a detailed rationale for conclusions
and recommendations, along with all comments provided on the test method. When a proposed test
guideline is submitted to the OECD based on a test method which has not undergone independent peer
review, the OECD should discuss and determine an appropriate responsible party to organize the necessary
independent review.
123.
Flexibility in the peer review process is considered to be essential. However care should be taken
to ensure that the peer review continues to be a balanced, expert, and fully open and transparent process.
Selection of Peer Reviewers
124.
The experts who serve as peer reviewers of validation studies should be technically competent,
credible and able and willing to perform their assessment and writing tasks. In addition, conflict of interest
criteria should be described. For example, some validation organisations require that the peer reviewer or
any immediate family member should not have financial or other conflict of interest with the test or theoutcome of the peer review. In order to ensure the integrity of the peer review process, the organisation
overseeing the peer review process should develop and apply specific criteria for assessing experts who are
candidates for the review. The following specific selection criteria could be considered:•

The experts have demonstrated expertise in one or more relevant scientific disciplines. The peer
review panel should include at least one expert from each of the identified disciplines. For certain
essential disciplines, it may be useful to have more than one expert on the review panel.

•

It is also important that some peer reviewers collectively have expertise in the design, conduct andevaluation of validation studies for new and revised toxicology tests and, when appropriate, that
one or more reviewers have understanding of animal welfare issues and the principle of the 3Rs ofanimal welfare.

•

It is also critical that peer reviewers are independent and not subject to any conflict of interest,
either in terms of previous substantial involvement in the process or of material interest in theoutcome of the review, e.g., financial. Alternatively, any particular interest in the outcome should
be declared at the start of the peer review process and the panel, as a whole, should be balanced
and independent.

125.
A panel of peer reviewers may be established for each validation project separately, as is done by
ICCVAM (60) and ECVAM, the US National Academy of Sciences and, on a case-by-case basis, in theOECD, or an established independent expert panel could be augmented with appropriate expertise. The
44

ENV/JM/MONO(2005)14
mechanism for nomination/selection of the peer reviewers may vary with the organization responsible fororganizing the review. Decisions on the mechanism to be used are made on a case-by-case basis. In allcases, nominations of qualified experts should be broadly solicited from appropriate scientific andstakeholder communities. It is crucial that the panel as a whole is balanced and that the peer review process
is open and transparent, especially if stakeholders or experts that have been involved in the validation are
members of the peer review panel. To allow for wide global acceptance of the validated test method,organisations or countries that have the task of arranging an independent evaluation of the validation
process should consider whether the peer review panel should be international in composition.
Charge to Peer Reviewers
126.
To facilitate the peer review process, the peer review panel could be provided a list of scientificallydirected questions appropriate for the nature of the test method being evaluated, however, the list should
not restrict the scope of the peer review. These questions could serve as a template for the peer review
panel’s evaluation. The questions are usually of a standard nature; e.g., has the test method data beencollected using studies designed and conducted to comply with appropriate international standards for
GLP? Other questions may be of a more specific nature to assess the appropriateness of the protocolcomponents with respect to attaining the objective of the proposed test method. The organisation
overseeing the peer review process should have the responsibility for developing questions to the reviewers
to ensure that all criteria are assessed by the panel
127.
The charge to the peer reviewers needs to pose searching and insightful questions that will elicit
input on the usefulness and limitations of the critical aspects of the test method for its specific proposed
use. The reviewers should provide comprehensive reviews on the extent to which each of the validation
criteria have been addressed, and their views on the proposed standardized protocol and decision criteria.
The panel should identify the chemical and physical properties of chemicals and products for which thetest method is likely to be useful and those for which it has not been adequately evaluated, or not shown to
be useful. Finally, the panel should comment on the appropriateness of the test performance (i.e., its
accuracy and reliability) for its intended purpose. The review should be complete, objective, and credible.
Peer Review Process128.
As described above, the first step of the peer review is provision of a complete test method
submission package to the peer reviewers. This package could include the charge, the validation study
reports and other documents that are to be evaluated by the reviewers, as well as other relevant documentsthat are to be considered by the reviewers, in responding to the charge.
129.
Whenever possible, all materials reviewed by the peer review panel should be made available toappropriate stakeholders. Stakeholder comments should be provided to the members of the peer review
panel for their consideration. In some Member countries there is a requirement to include the public in thiscommenting process. Ideally, it is desirable the peer review process be open and transparent. Some
validation agencies address this by having the peer review panel convene in public sessions. Practicalconsiderations, particularly for international reviews, should determine the best process for the peer review
panel. At the end of the review process, the overall assessment of the usefulness and limitations of the test
method should be included in the final peer review panel report. This final assessment will be based on thequestions posed to the panel.
130.
Peer reviews do not necessarily require full consensus agreement on each issue. However, adescription of any dissenting views and their supporting rationales should be provided. The results of thedeliberation by the peer review panel should be available in a written report subsequent to the final
recommendation of validation status of the test method by the panel. The peer review report should not45ENV/JM/MONO(2005)14
only discuss the usefulness and limitations of the proposed test but should provide recommendations for
future actions concerning the test method.
131.
The test method validation sponsor has the ultimate responsibility to approve the recommendations
from the peer review panel and decide whether the test method has been validated for its intended purpose
and should be recommended for regulatory acceptance. The sponsor of the test method will also be theresponsible party to address critical issues during the peer review e.g., how to address views fromindividual members of the panel and how to deal with the results from under-performing laboratories.
132.
Ideally, the peer review report should be available for widespread public dissemination, and thereport, or a synopsis of the report, be published in a scientific peer-reviewed journal. Ultimately, eachregulatory authority will have to make its own decision whether or not to accept a validated test method issuitable for its purpose.
133.
In certain instances, a previously reviewed test method which has been subjected to further
revision or development may be submitted for an additional or subsequent review of its validation status.
The level of effort devoted to this subsequent validation review should be commensurate with the degree
and importance of changes that have occurred to the protocol components of the test method.

46

ENV/JM/MONO(2005)14

VI. INTERNATIONAL REGULATORY ACCEPTANCE OF VALIDATED TESTS
134.
To conserve resources and time a test method can undergo validation studies and peer-review
evaluation prior to its consideration for an OECD Test Guideline. However, this is currently not arequirement in the process for developing OECD Test Guidelines.
135.
In a research and development setting, toxicology test procedures are conducted during the normal
course of the research, and the scientist performing the procedure, and those using the data, have sufficientknowledge of and experience with the procedure to make decisions based upon the data. Validated test
methods are reliable indicators of toxicological effects in the regulatory environment where test results areused to support the decision-making process.
136.
The regulatory acceptance process has generally been on a case-by-case basis, and regulatory
authorities have the option to accept results generated using a test method that has not undergone what
today would be considered formal validation (e.g., methods used in mechanistic studies that could help
underpin or explain results derived from other tests). However, acceptance of a test method by a specificregulatory authority does not necessarily indicate universal acceptance by other authorities. Acceptancepolicies differ from country to country and even, at times, among regulatory authorities within a country.
137.
Validation as described in this Guidance Document contributes strongly to the international
acceptance of any proposed test method and encourages and supports worldwide Mutual Acceptance ofData (MAD) (61). However, regulatory authorities may still have additional questions on the test method
beyond its established reliability and relevance, which could affect its regulatory acceptance.
Harmonisation of international regulatory acceptance of adequately validated test methods may beachieved by considering the guidance provided in this document. The regulatory acceptance of tests thathave not been subjected to prevailing validation processes is discouraged. In cases in which validation is
not considered necessary or appropriate, a written justification should be available.
Validation Study Outcomes
138.
Following adequate validation studies demonstrating the utility and the applicability of a test
method, it may be considered for adoption by regulatory agencies. A validated test method may besubmitted to the OECD for formal adoption as an OECD Test Guideline.
Criteria for Regulatory Acceptance139.
After a test method has undergone formal validation and is considered acceptable for specificproposed uses, a recommendation may be made that it be adopted as an OECD Test Guideline. As
mentioned earlier in this document, regulatory acceptance would be greatly facilitated by the involvement,
as early as possible in the validation process, of the regulatory agencies to which test results derived fromthe validated method will be submitted. In considering the regulatory acceptance of a new test, theprinciples and criteria described in Table 2 are important.

47

ENV/JM/MONO(2005)14

TABLE 2: PRINCIPLES AND CRITERIA FOR REGULATORY ACCEPTANCE
OF A NEW OR UPDATED TEST METHODa)

The submitted test method and supporting validation data should have been
subjected to a transparent and independent peer review process.
The peer review should be made by experts in the field, not substantively involved in
the development or validation of the test, knowledgeable of the method and data
evaluation, and not materially or financially affected by the outcome of the review.
Selection of peer reviewers shall be based upon scientific qualifications, expertise andexperience, regardless of employer or affiliation.

b)

Data generated by the test method should adequately measure or predict theendpoint of interest. For replacement test methods, the data should show alinkage between the proposed test method and an existing test method, and/or the
proposed test method and effects in the target or model species.

c)

The test method should generate data useful for hazard/risk assessment
purposes.
New test methods may fill a recognised data gap. The test method may be usefulalone or as part of a test battery or tiered testing approach. For substitute test methods
the generated data should be at least as useful as, and preferably better than, thoseobtained using existing test methods.

d)

The submitted test method and supporting validation data should adequately
cover a spectrum of chemicals and products representative of those administeredby the regulatory programme or agency for which the test method is proposed,
and the applicability and limitations of the test method should be clearly
described.

e)

The test method should be sufficiently robust (relatively insensitive to minorchanges in the protocol) and transferable among properly equipped laboratories
with adequately trained staff.
The test method preferably allows for standardisation. If highly specialised equipment,material, or expertise is required, means should be provided to facilitate test method
transferability.

f)

The test method should be time and cost effective and likely to be used in a
regulatory context.

g)

Justification should be provided (scientific, ethical, economical) for the new or
updated test method in light of already existing test methods.
Where appropriate, animal welfare concerns including the principle of the 3Rs ofreplacement, reduction and refinement, should be addressed.

48ENV/JM/MONO(2005)14140.
In a number of OECD member countries, a new or revised test can be adopted for use if it meets
three criteria, namely;•

It has been sufficiently validated; and,•it provides at least as much scientifically credible information as an existing test while using fewer
or no animals or while causing less animal suffering (7).

•It improves the safety assessments for man and the environment.

In such cases the principles and criteria listed for both validation and regulatory acceptance should be
fulfilled.
From Protocol to Test Guideline
141.
Based on past experience, regulatory bodies will routinely seek test procedures that provide forboth accuracy and flexibility in fulfilling testing requirements for regulatory purposes. This may result in
the use of a general international Test Guideline (TG) rather than a specific test method protocol.
142.
It is suggested that an expert group with expertise with the test method draft the Test Guideline.
The expert group should be able to distinguish between specific elements that it believes are critical to thegeneration of valid test results and, therefore, should be required in the Test Guideline, and those items thatcan be left to professional judgment and are, therefore, discretionary. If members of the expert groupcannot agree on whether a parameter should be a required or discretionary element, it would be advisable
to conduct a study to determine the impact of variations in that parameter on test results. It is
recommended that the expert group develop the Test Guideline after the test protocol is optimized,
standardized and validated.

49ENV/JM/MONO(2005)14

VII. NEW TEST SUBMISSIONS: SUPPORTING DOCUMENTATION
143.
When a new test method is considered ready for proposal as an OECD Test Guideline, submission
to the OECD Secretariat may be done by any of the following processes:i.

A Member country through its National Co-ordinator;ii.

the EU through the EC;iii.

an industry association through BIAC to the OECD;iv.

invited experts via a National Co-ordinator.

144.
Details of the process for adopting a test method as an OECD Test Guideline are provided in a
separate Guidance Document (4).
145.
As a practical example of how to organize information necessary to substantiate the validity of atest method proposed for regulatory acceptance consideration, material from the guideline for “theNomination and Submission of New, Revised, and Alternative Test Methods” by ICCVAM has been cited(60). This guideline, which is applicable to ICCVAM (the focus of which is the validation of alternative
methods) and its 15 member federal agencies, should not be understood as a binding standard for otherparties, but serves as an example found to be an effective and efficient way in one OECD Member country
to assemble and organize the necessary information and data to facilitate assessment of the validation
status of a test method according to established OECD or other validation criteria. Components of such a
submission follow.
Introduction and Rationale for the Proposed Test Method146.
For purposes of a new test submission, a description should be provided of how the proposed test
method can be used in the context of current or anticipated regulatory applications (e.g., as a screen in atiered testing strategy, as an adjunct test to provide mechanistic information, as a substitute or replacement
for an existing test method). The mechanistic basis of the proposed test method and the context in which it
will be used to measure or predict the toxicological activity of a test material or substance should be
discussed, as well as what is known about the similarities and differences of modes and mechanisms ofaction in the test system compared to the species of interest (e.g., humans for human health-related toxicity
testing). If applicable, the extent to which the proposed test method meets the performance standards of amechanistically and functionally similar validated and accepted test method should be addressed. The
sponsor should indicate the relevant classes of chemicals and products that can and cannot be evaluatedusing the proposed test method and any known limitations of the test method. Finally, the sponsor should
indicate where and how the proposed test method might be included in the overall safety or hazard
assessment process. In particular, if the proposed test method is part of tiered or battery approaches, theweight given to the new method relative to other tests in the tier or battery should be addressed.
Test Method Protocol Components
147.
The sponsor should use this section to explain and describe the basis for decisions on critical
functional, structural, and procedural elements of the test method protocol (a complete, detailed protocol
for the proposed test method should be provided in an appendix to the submission). This would include a
description of the extent to which the proposed test method protocol is similar to the protocol of a validated
mechanistically and functionally similar test method for which performance standards exist. The basis andimpact of any protocol modifications made during the validation of the proposed test method should be
discussed. The technical parameters of the proposed test method (e.g., vehicles, exposure time), the nature50ENV/JM/MONO(2005)14of the response evaluated, and the basis for proposed concurrent controls should be described. Concurrent
controls (negative, solvent, and positive), as appropriate, serve as an indication that the test method is
operative under the test conditions and provide a basis for experiment-to-experiment comparisons; they areusually part of the acceptance criteria for a given experiment. The acceptable ranges for the control
responses and historical data used to establish the acceptable range should be included.
148.
The nature of the data to be collected, the methods used for data collection, the type of media in
which data are stored, measures of variability, the statistical or non-statistical methods used to analyze andevaluate the data (including methods used to explore a dose-response relationship), and the decision
criteria (and their rationale) used to classify the response as positive or negative, if applicable, should be
described. The criteria for dose selection and the number of animals required, if any, for dose selection
and for the actual test should be stated. Both statistical and non-statistical methods used for data
evaluation should be justified. Any confidential information associated with the proposed test methodshould be indicated clearly; however, the inclusion of confidential information is discouraged.
149.
The number of replicate and/or repeat experiments needed to ensure an adequate study should be
provided, and the basis for the study design should be described. If replicate or repeat experiments are notpart of the proposed test method protocol, a rationale should be provided.
150.
The basis for selection of the proposed test method system should be provided. If an animal modelis used, the rationale for such use should be provided along with the rationale for selecting the species,
strain or stock, sex, acceptable age range, diet, frequency of dosing, the number of doses, and otherapplicable protocol components should be included.
151.
If the test method employs proprietary components, the procedures used to ensure their integrity(in terms of reliability and accuracy) from lot-to-lot and over time should be described. Also, proceduresthat the user may employ to verify the integrity of the proprietary components should be described.
Characterisation and Selection of Substances Used for Validation of the Proposed Test Method
152.
Since a test method may be determined to be effective for the evaluation of only certain classes ofchemicals rather than all types of chemicals and products, it is important to provide information regarding
those chemicals/classes which were used in the validation study. The rationale for the numbers and typesof substances tested during the validation process should be described. The specific chemical or
formulation names and relevant chemical and product classes for the substances tested should be specified.
Additional consideration should also be given to the fact that not all data sets will be homogeneous for agiven chemical characteristic (e.g., water solubility). In such cases, it may be useful to separate the data set
into smaller, more uniform subsets for data analysis based on physical-chemical properties or chemicalclass. To the extent possible, the following information should be provided for each test substance:•

Chemical Abstracts Service Registry Number (CASRN)•

Physical and chemical characteristics•

Solubility (e.g., Kow)•

Concentrations tested•

Purity•Source•Stability of the test substance in the test medium

51ENV/JM/MONO(2005)14
153.
Any characteristics of the test chemicals that may have direct impact on test method accuracy
and/or reliability should be described. Information regarding the use of coded substances and blind testing
during the validation process should be included. In the case of mixtures, the constituents and their relative
concentrations should be stated whenever possible. For a proposed test method mechanistically andfunctionally similar to a validated test method with established performance standards, the extent to which
the reference chemicals recommended in the performance standards were tested in the proposed test
method should be discussed, and any deviations from this list should be justified. In situations where alisted reference chemical is unavailable, the criteria used to select a replacement chemical should be
described. To the extent possible, when compared to the original reference chemical, the replacement
chemical should be from the same chemical/product class and produce similar effects in the in vivo
reference test method. In addition, if applicable, the replacement chemical should have been tested in thecomparable validated test method. If fewer than the listed reference chemicals are used, a rationale should
be provided. In addition to a written description of the substances tested, it is desirable that the information
be presented in tabular format, such as that depicted in Table 3. It is preferable that this information beprovided in both printed and electronic formats.
Table 3. Characterisation of Substances Tested.
Chemicalor
Product
Name

CASRN

ChemicalClass

Product
Class

Concentrations
TestedPuritySupplier or
Source of
Substance

Physical andChemicalCharacteristics

In Vivo Reference Data Used to Assess the Accuracy of the Proposed Test Method154.
If the proposed test method is intended to replace or substitute for an existing in vivo reference test
method, then a comparison of data between the proposed test method and the in vivo reference test method
is necessary. Data available from the target species for the biological or toxicity endpoint of interest
should also be provided. The submission should include:•

Comparative data for the same substances tested using the in vivo reference test method and, whereavailable and applicable, from human or other target species studies. If possible, individual animaland human data should be provided.

•

The criteria used to select the in vivo reference test method (or human) data.

•

The source of the in vivo reference test method data (e.g., the literature citation for publishedinformation, the laboratory study director, the year generated for unpublished data).

•

A description of the protocol(s) employed to generate the in vivo reference test method or humandata. Any modifications to the in vivo reference test method protocol(s) should be stated clearlyfor each data set, along with a discussion of the potential impact of these modifications on theassessment of the accuracy of the proposed test method.

•

A description of the quality of the in vivo reference test method data, including the extent of GLP
compliance (45)(64)(65)(66)(67)(68) and the use of coded test chemicals.

•

The original study data for the in vivo reference test method studies or references as to theiravailability.

52

ENV/JM/MONO(2005)14•

A summary of the availability and use of other, relevant toxicity information from the species ofinterest (e.g., data from human studies, accidental exposures for human health-related toxicity test
methods, results of post-marketing surveillance).

Test Method Data and Results
155.
The data generated by testing substances using the proposed test method protocol are reported inthis section. Any protocol modifications made during the development process and their impact should bestated clearly for each data set. All data, both original and derived, should be submitted, along with eachlaboratory’s summary judgment regarding the outcome of each study. The submission should include data
(and explanations) from all studies, whether successful or not. The statistical approach used to evaluate thedata should be described and justified.
156.
It is also important to describe the lot-to-lot consistency of the test chemicals, the time-frame of thevarious studies, and the laboratory(ies) in which the studies were conducted. A coded designation for eachlaboratory involved in an intra-laboratory evaluation of test method reliability and accuracy is acceptable.
Any original data not submitted should be available for review, if requested.
157.
Results should preferably be presented in tabular form for easy comparison of results from thereference test methods with those from the proposed test method. Table 4 depicts a suggested tabularformat for presenting the results, and can be used for presenting the information used in the accuracy
assessment. This information should be provided in both printed and electronic formats.

Table 4. Test Method Accuracy Assessment.
Chemicalor
Product
Name

CASRN

ChemicalClass

Product
Class

Result Using
Proposed
Test Method(quantitative)

ResultUsing
Proposed
Test
Method(+/-)

Result Using
Reference
Test Method(quantitative)*

ResultUsing
Reference
Test
Method(+/-)

References
or
Data
SourcesComments*Where possible, data from the in vivo reference test method should be separated into single columns for eachspecies with available information. Human data should be always presented independently of nonhuman data. If
applicable, corresponding data obtained using the mechanistically and functionally similar validated test method withestablished performance standards should be provided.

Test Method Relevance (Accuracy)
158.
This section should describe the accuracy (e.g., sensitivity, specificity, positive and negative
predictivity, false positive and false negative rates) of the proposed test method and it should be comparedto that obtained for the reference test method currently accepted by regulatory agencies and also compared
to data or recognized toxicity information from the species of interest (e.g., humans for human-healthrelated toxicity testing). In instances where the proposed test method is measuring or predicting an
endpoint for which there is no pre-existing test method, the predictions should be compared to relevant
information from the species of interest in order to determine the accuracy of the responses. In cases
where the proposed test method is mechanistically and functionally similar to a validated test method withestablished performance standards, the accuracy of both test methods should be compared. When the
results obtained using the proposed test method is discordant from that obtained using the comparable
validated test method, the frequency of correct predictions of each test method compared to available

53

ENV/JM/MONO(2005)14
toxicity information from the species of interest should be presented. The basis for any discordance inresults for the following comparisons should be discussed:•

The proposed test method and currently accepted reference test methods.

•

The proposed test method and, if applicable, the comparable validated test method with establishedperformance standards.

•

The proposed test method and the accepted reference test method in predicting responses in thespecies of interest, where data from the species of interest is available.

159.
The submission should include a discussion of the strengths and limitations of the proposed test
method and should describe salient issues of data interpretation.
Test Method Reliability (Repeatability/Reproducibility)160.
An assessment of test method reliability (repeatability and reproducibility) should be provided.
This assessment should include discussion of the rationale for the selection of the substances used toevaluate intra- and inter-laboratory reproducibility, and the extent to which those substances represent therange of possible test outcomes. Outlying values should be identified and discussed. A quantitative
statistical analysis of the extent of intra- and inter-laboratory variability, such as that described in ASTM
Publication Number E691-92 (62) or coefficient-of-variation analysis should be included. Measures ofcentral tendency and variation should be summarized for historical control data (negative, vehicle, andpositive, where applicable). In cases where the proposed test method is mechanistically and functionallysimilar to a validated test method with established performance standards, the reliability of the two test
methods should be compared and the potential impact of any differences discussed.
Test Method Data Quality
161.
The extent of adherence to national and international GLP guidelines (46)(63)(64)(65)(66)(67) for
the data presented in the submission, as well as the results of any data quality audits, should be included inthis section. Deviations from GLP guidelines and the impact of any non-compliance detected in audits
should be described. Information on the availability of laboratory notebooks and other data retained by thesponsor(s) for external audits should be stated. All data should be supported by relevant documentation inlaboratory notebooks.
Other Scientific Reports and Reviews
162.
In this section, the submission should discuss all data from other published or unpublished studies
conducted using the proposed test method. Comment should be provided on any conclusions arising froman independent peer-review of the results derived from and performance of the proposed test method
and/or from other scientific reviews of the proposed test method. The conclusions of such scientificreports or reviews should be compared to the conclusions reached in the submission. Any other ongoing orplanned evaluations of the proposed test method should be described. In cases where the proposed test
method is mechanistically and functionally similar to a validated test method with established performancestandards, the results of studies conducted subsequent to the evaluation should be included, and any impact
on the reliability and accuracy of the proposed test method discussed.
Animal Welfare Considerations (Refinement, Reduction and Replacement)
163.
A description should be included of how the proposed test method will refine, reduce, or replace
animal use as compared to currently employed methods used for the endpoint of interest. If the proposed54

ENV/JM/MONO(2005)14
test method requires the use of animals, the rationale for such use should be provided. A description of thesources used to determine the possible availability of alternative test methods that would refine, reduce, or
replace animal use for the endpoint of interest should be provided (68)(69). The description should include,
at a minimum, the databases searched, the search strategy, the search date(s), the database search results,
and the rationale for not utilizing available alternative methods. The basis for determining the appropriate
number of animals for the proposed test method should be described. If the test involves potential animal
pain and distress, the procedures and approaches that have been incorporated to minimize and, wheneverpossible, to eliminate the occurrence of such pain and distress should be discussed (10).
Practical Considerations
164.
In this section, the cost and time involved in conducting a study using the proposed test methodshould be specified and compared to the reference test method(s) and, if applicable, to the mechanisticallyand functionally similar validated test method with established performance standards. Also included inthis section should be:•

A discussion of the facilities and major fixed equipment needed to conduct the test method.

•

The general availability of other necessary equipment and supplies.

•

The required level of training, expertise, and demonstrated proficiency needed by the study
personnel.

References
165.

A listing of all publications referenced in the submission should be provided.

Supporting Materials
166.

Appendices should contain:•

A detailed protocol for the proposed test method•

Copies of all relevant publications, including those containing data from the proposed test method,
the in vivo reference test method, and if applicable, a comparable validated test method•

All available original data used to evaluate the validity of the proposed test method•

Suggested performance standards to be considered, if performance standards for the proposed test
method do not exist.

55

ENV/JM/MONO(2005)14

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Final Rule. 40 CFR Part 792. Available: [http://www.gpoaccess.gov/nara/index.html]
65.
U.S. FDA. (1999). Good Laboratory Practice for Nonclinical Laboratory Studies. 21 CFR Part 58.
Available: [http://www.gpoaccess.gov/nara/index.html]
66.
OECD (1998). Principles of Good Laboratory Practice and Compliance Monitoring.
ENV/MC/CHEM (98)17. Paris, France: Organisation for Economic Co-operation and Development.
Available: [http://www.oecd.org/]
67.
Cooper-Hannan, R, Harbell, J, and Coecke, S, (1999). The principles of good laboratory practice:application to in vitro toxicology studies. ATLA 27:539-577.
68.
USDA (1966). Animal Welfare Act and Regulations. 7 USC: 2131-2156. Available:[http://warp.nal.usda.gov:80/awic/legislat/usdaleg1.htm]
69.
Public Health Service. (1996). Public Health Service Policy on Humane Care and use ofLaboratory Animals. Washington, DC: U.S. Department of Health and Human Services. Available:[http://grants1.nih.gov/grants/olaw/references/phspol.htm]

61ENV/JM/MONO(2005)14

ANNEX I
DEFINITIONS AND GLOSSARY
The definitions presented here are to be considered working definitions for the purpose of this document
also giving a broader view of the variability of terminology used by different validation agencies andorganisations. All definitions and terminology will be consistent with the proposed OECD/IPCS
Harmonisation of Terminology.
3Rs (principle of): Reduction (of animal use); Refinement (to lessen pain or distress and to enhance animal
well-being); and Replacement (of an animal test with one that uses non-animal systems or phylogenetically lower species).
Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
with “concordance” to mean the proportion of correct outcomes of a test method.
Adjunct test: Test that provides data that add to or help interpret the results of other tests and provide
information useful for the risk assessment process
Alternative test: A test that: reduces the numbers of animals required; refines procedures to lessen or
eliminate pain or distress to animals, or enhance animal well-being; or replaces animals with non-animal
systems or with non-sentient species.
Applicability domain: A description of the physicochemical or other properties of the substances forwhich a test method is applicable for use.
Assay: Uses interchangeably with Test.
“Catch-up” validation study: A validation study for a test method that is structurally and functionallysimilar to a previously validated and accepted reference test method. The candidate test method shouldincorporate the essential test method components included in performance standards developed for thereference test method, and should have comparable performance when evaluated using the reference
chemicals provided in the performance standards.
Coded chemicals: Chemicals that are labelled by code when delivered to the laboratory for testing so thatthey can be tested without the laboratory personnel having knowledge of their identity or anticipation of
the test results. Coded chemicals are used to avoid intentional or unintentional bias when performing
laboratory tests or evaluating test results.
Concordance: This is a measure of test method performance for test methods that give a categorical result,and is one aspect of “relevance”. The term is sometimes used interchangeably with “accuracy”, and isdefined as the proportion of all chemicals tested that are correctly classified as positive or negative.
Concordance is highly dependent on the prevalence of positives in the types of substances being examined.
Data interpretation procedure (DIP): An interpretation procedure used to determine how well the resultsfrom the test predict or model the biological effect of interest. See Prediction Model.
Decision Criteria: The criteria in a test method protocol that describe how the test method results are used
for decisions on classification or other effects measured or predicted by the test method.
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Definitive test: A test that is considered to generate sufficient data to determine the specific hazard or lackof hazard of the substance without the need for further testing, and which may therefore be used to makedecisions pertaining to hazard or safety of the substance.
Dose response assessment: The second of four steps in risk assessment consisting in the analysis of therelationship between the total amount of an agent administered to, taken up or absorbed by an organism,
system or (sub)population and the changes developed in that organism, system or (sub)population inreaction to that agent, and inferences derived from such an analysis with respect to the entire population.
Endpoint: The biological or chemical process, response, or effect, assessed by a test.
Expert system: a computer-based tool that generates predictions of endpoints by applying (Q)SARs and/or
rules designed to recreate the reasoning of experts. Expert systems may also contain a database ofexperimental data which may be consulted directly and which may be used during the application of therules.
Exposure assessment: Analysis of the exposure to an organism, system or (sub)population of an agent(including its derivatives). Exposure Assessment is the third step in the process of Risk Assessment.
False negative: A substance incorrectly identified as negative or non-active by a test method, when in fact
it is negative or non-active.
False negative rate: The proportion of all positive substances falsely identified by a test method as negative.
It is one indicator of test method performance.
False positive: A substance incorrectly identified as positive or active by a test, when in fact it is negative
or non-active.
False positive rate: The proportion of all negative (non-active) substances that are falsely identified as
positive. It is one indicator of test performance.
Good Laboratory Practices (GLP): Regulations promulgated by a number of countries and national
regulatory bodies that describe record keeping and quality assurance procedures for laboratory records thatwill be the basis for data submissions to regulatory authorities. Also the subject of the OECD Series on“Principles of Good Laboratory Practise and Compliance Monitoring” (42).
Hazard: The potential for an adverse health or ecological effect. The adverse effect is manifested only ifthere is an exposure of sufficient level.
Hazard identification: The identification of the type and nature of adverse effects that an agent has asinherent capacity to cause in an organism, system or (sub)population. Hazard identification is the first stepin the process of Risk Assessment
Hierarchical (tiered) testing approach: An approach where a series of tests to measure or elucidate aparticular effect are used in an ordered sequence. In a typical hierarchical testing approach, one or a few
tests are initially used; the results from these tests determine which (if any) subsequent tests are to be used.
For a particular chemical, a weigh-of-evidence decision regarding hazard could be made at any stage (tier)in the testing strategy, in which case there would be no need to proceed to subsequent tiers.
In silico models: Approaches for the assessment of chemicals based on the use computer-based estimations
or simulations. Examples include structure-activity relationships (SAR), quantitative structure-activity
relationships (QSARs), and expert systems.
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Inter-laboratory reproducibility: A measure of the extent to which different qualified laboratories, using thesame protocol and testing the same substances, can produce qualitatively and quantitatively similar results.
Inter-laboratory reproducibility is determined during the prevalidation and validation processes, andindicates the extent to which a test can be successfully transferred between laboratories, also referred to as
between-laboratory reproducibility.
Intra-laboratory repeatability: The closeness of agreement between test results obtained within a single
laboratory when the procedure is performed on the same substance under identical conditions within agiven time period.
Intra-laboratory reproducibility: A determination of the extent that qualified people within the samelaboratory can successfully replicate results using a specific protocol at different times. Also referred to aswithin-laboratory reproducibility.
Lead laboratory: The laboratory selected to perform the initial development of a standardised andoptimised test method protocol and to train the other laboratory personnel in the protocol procedures forthe performance of an inter-laboratory validation study. This laboratory may also be used to produce thereference data against which the performance of the other laboratories will be evaluated.
“Me-too test”: A colloquial expression for a test method that is structurally and functionally similar to avalidated and accepted reference test method. Such a test method would be a candidate for catch-up
validation.
Mechanistic (based) test: A test that provides a direct relationship between the biological effects observed
and the biological effects of interest.
Modular approach: A general conceptual framework that combines the use of retrospective and prospective
approaches to validation. According to the ECVAM proposal, the information needed to support thevalidity of a test methods should be organised into seven modules.
Optimised test protocol: A test protocol that has been revised and improved based on the results obtainedin prevalidation and validation studies.
Partial replacement test: A test method that enables animal reduction by replacing an animal test for one ormore of its endpoints (but not all of them), a limited range of chemicals (but not all chemicals), or a limited
range of the response values. A partial replacement test can contribute to the complete replacement of an(animal) test when used in a complementary fashion in a (tiered) testing strategy.
Peer involvement: The interaction of outside experts of equivalent expertise and experience with theexperts performing the work during the development of the scientific product. Such interaction enables anopen exchange of ideas, data, and insights. Peer involvement can occur throughout the development of thescientific product and adds to the scientific credibility of a product.
Peer review: A documented critical review of a specific scientific work product or products, which is
conducted by experts who are independent of the experts who performed the original work but who arecollectively comparable in technical expertise.
Performance characteristics: The operational characteristics of a test are the measures of its performance
under specific conditions, including its reliability and accuracy, and are an indication of the test’s
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Performance standards: Standards, based on a validated test method, that provide a basis for evaluating thecomparability of a proposed test method that is mechanistically and functionally similar. Included are (1)essential test method components; (2) a minimum list of reference chemicals selected from among thechemicals used to demonstrate the acceptable performance of the validated test method; and (3) thecomparable levels of accuracy and reliability, based on what was obtained for the validated test method,that the proposed test method should demonstrate when evaluated using the minimum list of reference
chemicals.
Prevalence: The proportion of positive or negative substances in the types of substances of interest. In
practice, prevalence cannot be determined precisely, but can be estimated from a sample of substancestaken from the types of substances of interest.
Prevalidation: The initial phase(s) of a validation study. A small-scale study intended to obtain preliminary
information on the relevance and reliability of a test method. Based on the outcome of those studies, thetest method protocol may be modified or optimised to increase intra- and/or inter-laboratory reproducibility
and accuracy in subsequent validation studies
Prediction Model (PM): a formula or algorithm (e.g., formula, rule or set of rules) used to convert theresults generated by a test method into a prediction of the (toxic) effect of interest. Also referred to as
decision criteria. A prediction model contains four elements: (1) a definition of the specific purpose(s) for
which the test method is to be used; (2) specifications of all possible results that may be obtained, (3) analgorithm that converts each study result into a prediction of the (toxic) effect of interest, and (4)specifications as to the accuracy of the prediction model (e.g., sensitivity, specificity, and false positive andfalse negative rates). Prediction models are generally not used in in vivo ecotoxicological tests.
Predictivity (negative): The proportion of correct negative responses among substances testing negative by
a test method. It is one indicator of test method accuracy. Negative predictivity is a function of thesensitivity of the test method and the prevalence of negatives among the substances tested.
Predictivity (positive): The proportion of correct positive responses among materials testing positive by atest method. It is one indicator of test method accuracy. Positive predictivity is a function of thesensitivity of the test method and the prevalence of positives among the substances tested.
Proprietary test method: A test method for which manufacture and distribution is restricted by patents,
copyrights, trademarks, etc.
Prospective validation: An approach to validation when some or all information necessary to assess thevalidity of a test are not available, and therefore new experimental work is required.
Protocol: The detailed, unambiguous step-by-step description of a test method that directs the laboratory
as to how to perform the test method. The test method protocol includes the listing and description of all
preparations, reagents, supplies, and equipment needed, and all criteria and procedures for generating andevaluating test data.
(Q)SARs (Quantitative Structure-Activity Relationships): Theoretical models for making predictions ofphysicochemical properties, environmental fate parameters, or biological effects (including toxic effects inenvironmental and mammalian species). They can be divided into two major types, QSARs and SARs.
QSARs are quantitative models yielding a continuous or categorical result while SARs are qualitative
relationships in the form of structural alerts that incorporate molecular substructures or fragments related tothe presence or absence of activity.

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Quality assurance: A management process by which adherence to laboratory testing standards,
requirements, and record keeping procedures, and the accuracy of data transfer, are assessed by individuals
who are independent from those performing the testing.
Reference chemicals: Chemicals selected for use in the validation process, for which responses in the invitro or in vivo reference test system or the species of interest are already known. These chemicals should
be representative of the classes of chemicals for which the test method is expected to be used, and should
represent the full range of responses that may be expected from the chemicals for which it may be used,from strong, to weak, to negative. Different sets of reference chemicals may be required for the different
stages of the validation process, and for different test methods and test uses.
Reference data: An agreed-upon set of values against which the values obtained using the new test will becompared.
Reference species: The species used in the reference or traditional test to which a new or revised test is
being compared. This may be the species of interest, or it may be a surrogate species when it is notpossible to perform testing on the species of interest (e.g., humans).
Reference test method: A test method against which the results from the new test method are being
compared.
Regulatory acceptance: The formal acceptance of a test method by regulatory authorities indicating that thetest method may be used to provide information to meet a specific regulatory requirement.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method.
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability.
Repeatability: The agreement among test results obtained within a single laboratory when the procedure is
performed on the same substance under identical conditions. (see Reliability)
Replacement test: A test which is designed to substitute for a test that is in routine use and accepted forhazard identification and/or risk assessment, and which has been determined to provide equivalent or
improved protection of human or animal health or the environment, as applicable, compared to t theaccepted test, for all possible testing situations and substances.
Reproducibility: The agreement among results obtained from testing the same substance using the sametest protocol. (see reliability)
Retrospective validation: An assessment of the validation status of a test method carried out by considering
all available information and data generated from one or more validation studies.
Ring test: A multi-laboratory validation study in which all laboratories test the same substances using
identical test protocols, often used for eco-toxicity test method validation. The purpose of the study is todetermine inter- and intra-laboratory reproducibility of a test method. Sometimes used interchangeably
with “round-robin test”.
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Risk: The probability or degree of concern that a defined exposure to a substance will cause an adverse
effect in the species of interest or in the environment. Risk is determined by assessing hazard identification,
dose-response, and exposure information.
Risk assessment: A process intended to calculate or estimate the risk to a given target organism, system or(sub)population , including the identification of attendant uncertainties, following exposure to a particular
agent, taking into account the inherent characteristics of the agent of concern as well as the characteristicsof the specific target system. The Risk Assessment process includes four steps: hazard identification,
hazard characterisation (related term: dose-response assessment), exposure assessment, and risk
characterisation. It is the first component in a risk analysis process. The definition of risk assessment mayvary between Member countries.
Robust(ness): The insensitivity of test results to departures from the specified test conditions when
conducted in different laboratories or over a range of conditions under which the test method might
normally be used. If a test is not robust, it will be difficult to use in a reproducible manner within andbetween laboratories.
SAR: See (Q)SARs.
A screen/screening test is often a rapid, simple test method conducted for the purpose of classifying
substances into a general category of hazard. The results of a screening test generally are used forpreliminary decision making in the context of a testing strategy (i.e., to assess the need for additional andmore definitive tests). Screening tests often have a truncated response range in that positive results may beconsidered adequate to determine if a substance is in the highest category of a hazard classification system
without the need for further testing, but are not usually adequate without additional information/tests tomake decisions pertaining to lower levels of hazard or safety of the substanceSensitivity: The proportion of all positive/active substances that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method.
Specificity: The proportion of all negative/inactive substances that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results and is an important consideration inassessing the relevance of a test method.
Standard Operating Procedures (SOP): Formal, written procedures that describe in detail how specificroutine, and test-specific, laboratory operations should be performed. They are required by GLP.
Substitute test: A new or revised test that is proposed for use in lieu of a currently used test, regardless ofwhether the test is being used as a screen, definitive test, or adjunct test, but which may not be able tocompletely replace the current test method for all testing situations and all substances.
Surrogate: A test species or system used in the place of another target species or test system.
Target species: The species for which information on the potential toxicity or effects of a substance issought.
Test (or assay): An experimental system used to obtain information on the adverse effects of a substance.
Used interchangeably with assay.

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Test battery: A series of tests usually performed at the same time or in close sequence. Each test within thebattery is designed to complement the other tests and generally to measure a different component of amulti-factorial toxic effect. Also called base set or minimum data set in ecotoxicological testing.
Test method development: The research process before validation in which a test protocol is developedand standardized. The process of test method development should ideally lead to a protocol which is
sufficiently detailed and comprehensive to enable the test to undergo prevalidation.
Test method: A process or procedure used to obtain information on the characteristics of a substance oragent. Toxicological test methods generate information regarding the ability of a substance or agent toproduce a specified biological effect under specified conditions. Used interchangeably with “test” and“assay”.
Test method sponsor: An entity that has proposed a test method for incorporation into a Test Guideline,
and for eventual regulatory consideration and acceptance. The test sponsor is usually responsible fororganising and providing all the available, relevant information to the organisation overseeing or reviewing
the validation status of the test method, or to the agencies which are being asked to accept the test.
Tiered testing approach: Testing which uses test methods in a sequential manner; the test methods selectedin each succeeding level are determined by the results in the previous level of testing.
Transferability: The ability of a test procedure to be accurately and reliably performed in independent,
competent laboratories.
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be foundacceptable for the proposed purpose.
Valid test method: A test method considered to have sufficient relevance and reliability for a specificpurpose and which is based on scientifically sound principles. A test method is never valid in an absolutesense, but only in relation to a defined purpose.
Validation: The process by which the reliability and relevance of a particular approach, method, process orassessment is established for a defined purpose.
Validation management group (VMG): The independent oversight group, consisting of individuals withexperience with the types of assays being performed, biostatisticians, and others with knowledge of the
purpose of the prevalidation and multi-laboratory phases of the validation study, which is responsible forapproving the design and implementation of the study and the selection of the participating laboratories,
and co-ordinating the evaluations of the study results. A VMG is also called Management Committee orManagement Team.

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ANNEX II
EXAMPLES OF VALIDATION STUDIES WITHIN DIFFERENT AREAS OF TEST METHODDEVELOPMENT
In this Annex, five examples of validation studies within different areas of test method development arepresented. These examples are intended to illustrate how different approaches have been used in thevalidation of test methods that have been proposed and accepted for regulatory use. The examples also
provides good reasons why the validation process should be kept flexible and on a case-to-case basis. One
goal of this Guidance Document is to present a common understanding of how validation in in vitro, invivo and ecotoxicity testing was addressed; and as a second goal, the Guidance Document presents andadvocates common validation principles while also recognizing distinctions that can be applied to any areaof toxicology, hazard identification/assessment, and risk assessment.
The ecotoxicology examples (1 and 2), for instance, describe a process with an emphasis on
standardization of a method rather than on determining the relevance and sensitivity of the method. By
including the actual species of interest, ecotoxicity tests incorporate endpoints which are intrinsically
relevant and their sensitivity inherent. Therefore, the elements of validation that should be demonstrated
are reduced primarily to those of reproducibility. Inter-laboratory (ring) testing functions to verify the
reproducibility of the test method and provide an empirical basis for standardizing a Test Guideline andestablishing its suitability for regulatory use.
The third example on in vitro tests for skin corrosion that have resulted in the acceptance of TGs 430 and
431 gives practical guidance on a variety of approaches to validation. Including the steps of prevalidationand validation, but also a special study on tiered testing approaches in addition to a catch-up validation
study.
The in vivo Local Lymph Node assay (example 4) represents an alternative test method to TG 406 offeringrefinement and the validation process is outlined. The fifth example (updated TG 425) is a replacement test
for the Acute Oral Toxicity Study (TG 401) where the testing is based on a staircase testing approach.
Example 1:
Example 2:
Example 3:
Example 4:
Example 5:

The OECD Lemna growth inhibition test. Development and ring-testing of draft OECD
Test Guideline.
Report of the Final Ring Test of the Daphnia magna Reproduction Test.
Validation, Special Study and “catch-up” Validation of In vitro Tests for Skin Corrosion
Potential – The Human Skin Model Test
The Local Lymph Node Assay (LLNA)
The Up-and-Down Procedure for Acute Oral Toxicity (TG 425)

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Example 1

The OECD Lemna growth inhibition test: development and ring-testing of a draft OECD Test
Guideline.

Environment Agency, Research and Development Technical Report EMA 003, 90 pp., by I. Sims, P.
Whitehouse, and R. Lacey, WRc plc (Contractor Report No. EA 4784), Henley Road, Medmenham,
Marlow, Buckinghamshire, UK (1999).
Funding provided by Environment Agency, Bristol, UK and USEPA Office of Prevention, Pesticides andToxic Substances, Washington, DC.

1. Introduction and Rationale for the Proposed Test Method Validated
In a review of Test Guideline (TG) requirements in 1995 (See DRP on Aquatic Testing Methods for
Pesticides and Industrial Chemicals No. 11, OECD Environmental Health and Safety Publications, Series
on Testing and Assessment, 1998) an aquatic higher plant toxicity test method was highlighted as a highpriority for development by the OECD. This led to an initiative by KEMI (Sweden) to develop a new Test
Guideline based on aquatic macrophytes of the genus Lemna, which was subsequently taken forward by
WRc (UK). Inhibition of growth is measured (as changes in yield or biomass and in growth rate) in these
floating vascular plants over a period of seven days.
An expert panel or workgroup (steering group) was established in early 1997. Workgroup meetings were
held in March and November 1997. At the first meeting research needs that would enable a new OECD
TG to be written, were identified and discussed. A “Phase 1", or prevalidation study, was then conducted in
1997 and 1998 by members of the panel to fill in these needs or knowledge gaps. Areas addressed
included choice of test species, test media, endpoints, and data analysis. Results of Phase 1 were discussed
at the second panel meeting, and work on a draft OECD TG began. Plans for “Phase 2,” an internationalring-test or validation study were discussed. A draft test method (June 1998) was then developed and thiswould form the basis for the method to be validated in Phase 2. The ring-test was conducted in 1998-1999,
with financial support provided by the UK Environmental Agency and the US EPA, Office of Prevention,
Pesticides and Toxic Substances. A final report was completed later in 1999. A draft of this report had
been reviewed by members of the panel. No formal peer review of the ring-test was conducted.
Another draft of this method was developed by the OECD Secretariat in October 2000, with approval by
the panel, and distributed to Member countries for comment. Objections were raised by some Member
countries on the test endpoints and statistical analysis sections of the draft. Resolution of these objections
delayed approval of the proposed new TG. None of these objections had anything to do with aspects ofvalidation or peer review. Discussions and commenting on several more draft TG’s then occurred. Afteran expert consultation meeting were held in October 2003, agreement among experts from Member
countries was achieved and this TG will appear in 2004 as TG 221 in the 16th Addendum of the OECD
Test Guidelines Programme.
The TG 221 will be used to assess the effects of pesticides and industrial chemicals on floating aquatic
vascular higher plants, and will play an important role in regulation of these substances. It will serve as animportant supplement to algal tests, and a Lemna test will be a useful addition directly applicable in tiered
testing schemes used by Member countries for hazard identification/assessment and risk assessment.
Exactly how this TG will be used in regulatory processes will be left to each Member country to decide.

70ENV/JM/MONO(2005)14
2. Test Method Protocol Components
The principle of the test method is as follows. Exponentially growing plant cultures of the duckweed
genus Lemna are allowed to grow as monocultures in different concentrations of the test substance over a
period of seven days. The objective of the test is to quantify substance-related effects on vegetative growthover this period based on assessments of selected measurement or response variables (frond number, andone other variable for yield such as total frond area, dry weight or fresh weight). To quantify substancerelated effects, growth in the test solutions is compared with that of the controls and the concentration
bringing about a specified x% inhibition of growth (e.g., 50%) is determined and expressed as the ECx(e.g., EC50). In addition, the lowest observed effect concentration (LOEC) and the no-observed effectconcentration (NOEC) may be statistically determined.
The method followed in the ring-test was the draft OECD “Lemna growth inhibition test planned ring-test”,
1998, Appendix C in the Environment Agency Technical Report cited above. This method will not be
discussed in detail here. As a result of the ring-test a revised draft was written (draft OECD Lemna growth
inhibition test (JUNE 1999)), which is the Appendix B of the Technical Report. Current, complete details
on this method, with subsequent changes made since the 1999 draft, are given in the OECD publication:
OECD Guidelines for the Testing of Chemicals – Revised proposal for a new Guideline 221: Lemna sp.
Growth inhibition test [ENV/JM(2004)33/ANN4], October 22, 2004, and also will not be repeated here.
3. Substances Used for Validation of the Proposed Test Method
After considerable debate among the steering group members, two chemicals were chosen for use in thering test: potassium dichromate and 3,5-dichlorophenol. These chemicals were selected because:
1) Their effects on the growth of Lemna are well-documented.
2) They have different modes of toxic action, with low-moderate toxicity so that use of higher
concentrations helps to minimize errors in preparation of solutions and analysis.
3) Stock solutions could be prepared without the use of solvents, and water solubility is “wellabove” toxic concentrations.
4) They are stable over the test period of seven days, and hydrolytically stable over several months(stock solution).
5) Low volatility from water, and stable/”conservative” speciation over the pH range recommended
for the test.
6) Ease of analysis and readily available to all ring-test participants.
7) They represent two well-defined groups of toxicants, i.e., metals and organics, with a “reasonably
well-defined” dose-response.
Both chemicals have been widely used in ring-tests and as reference toxicants in tests with other organisms.
Since the dose-response appears to be “fairly shallow” the selection of test concentrations was especially
important. The test concentrations were chosen to bracket the EC50 with an interval between
concentrations not to exceed x3.2.
4. In Vivo Reference Data used for an Assessment of the Accuracy of the Proposed Test Method(where appropriate)
A reference test method was not used for comparisons because the Lemna test is a new test method, which
was based on a composite of several existing methods used by different Member countries. While test data71

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were available from each of these national test methods there was great uncertainty on which data to use,such that it was found to be impractical to make these comparisons. In the ring-test, results from thecontrols were compared with the treatments. Performance characteristics assessed included the extent towhich validity (doubling time) criteria were met, the intra-laboratory repeatability (used to describe theprecision of the method), and the inter-laboratory reproducibility (used to assess the accuracy with which
toxicity is estimated). Thirty-seven laboratories from 15 Member countries participated in the ring-test.
5. Test Method Data and Results
The 37 participating laboratories were allowed to test two species of duckweed, Lemna minor or Lemna
gibba. More of the laboratories submitted data for the first species than for the second species, andpotassium dichromate was used more extensively in the ring-test than was 3,5-dichlorophenol. Many
laboratories repeated tests with the same toxicant and species. Adherence to the 1998 draft TG was good.
All participants supplied EC50 data calculated from average specific growth rates as based on frond
number, as required by the draft TG, and most (83%; n=191) met the test acceptability criterion for control
doubling time. Those tests that failed this criterion were usually conducted under conditions of low
illumination or low temperature, or both. Few exceedences of water quality criteria occurred, although themost common problem was an increase in pH in the control medium that was greater than that advised in
the 1998 draft. However, this did not seem to be associated with reduced growth rates.
The ranges of measured EC50 values for the two species of Lemna and the two test substances are
summarized:
Inter-quartile ranges of EC50 values generated in the Lemna inter-laboratory ring-test
Test Substance

Lemna minor (mg/L)

Lemna gibba (mg/L)

Potassium dichromate

2-4 (n=61)

8-30 (n=28)

3,5-dichlorophenol

2.7-3.4 (n=52)

6.00-7.00* (n=4)* = range of reported EC50 values.

These data were analyzed to identify the contributions to variability in EC50 values resulting from intra-,and inter-laboratory variability. Possible sources of variability were partitioned by using Residual
Maximum Likelihood (REML) procedures, with log-transformed EC50 data. Those EC50 values fromtests where the doubling time for control frond number was exceeded or where the EC50 was extrapolated
beyond the concentration range tested were excluded from the analysis. Both sources of variability weregreater in experiments using potassium dichromate but the inter-laboratory variability generally accounted
for a greater proportion of the observed variability than the intra-laboratory variability. In tests with L.
minor and potassium dichromate, two laboratories were responsible for a high proportion of the interlaboratory variability, suggesting their EC50 estimates were “rather extreme.”
Participating laboratories obtained their own potassium dichromate or 3,5-dichlorophenol, or both, andprepared their own stock solutions for these chemicals.

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6. Test Method Performance (Accuracy)
Comparisons with a reference method were not made since in ecotoxicological testing there is generally no
accepted reference value. The closest to a reference value in ecotoxicology is where a weight-of-evidence
approach is taken. Ecotoxicity test values derived from testing methods with “similar” species may cluster
together to give a mean toxicity value. A value from another method may fall within this cluster. Thus,
this approach may give some crude indication of method accuracy.
Accuracy has been defined by ICCVAM as determinations of concordance, sensitivity, specificity, positive
and negative predictivity, and false positive and negative rates. These various determinations were notmade in the Lemna ring-testing.
7. Test Method Reliability (Repeatability/Reproducibility)
Quality control criteria were derived and expressed in terms of the deviation from the “consensus” mean
EC50 (the geometric mean of the mean EC50s obtained by each laboratory). Estimates of inter-laboratory
variance were calculated. When used in conjunction with reference toxicant data, these criteria may be
used by laboratories to assess the accuracy with which they perform Lemna growth inhibition tests. They
may also be used by regulatory authorities when assessing the quality of data to be used in hazard
identification/assessment and in risk assessment of chemicals, so that excessive bias and variability can be
identified. For EC50 values in tests using L. minor and potassium dichromate, the recommended accuracy
criteria are 0.6-7.2 mg/L and for this species and 3,5-dichlorophenol, the criteria are 1.7-5.7 mg/L. These
ranges serve as “targets” for judging accuracy of L. minor tests with potassium dichromate.
Quality control criteria could not be established for Lemna gibba because there was a lack of suitable data.
Considerably fewer laboratories performed ring-tests with this species (see table above). Because of thislack, very wide control limits would be calculated.
Quality control criteria can also be derived for precision, expressed as the range of EC50 values generated
within a laboratory. Estimates of inter-laboratory variability are used, and REML-analysis provided an
estimate of the underlying repeatability of duckweed growth inhibition tests for the two toxicants. A seriesof tests with the reference toxicant should be performed. The variance of the EC50 values from the seriesof repeat tests is compared with the underlying variance that would be expected, based on the ring-test.
This comparison may be done using a chi-square test and an appropriate significance level. Chi-squares
were determined for different numbers of repeat tests, based on a given significance level, and multipliervalues were calculated. If the variance of EC50 values from a series of tests in a laboratory exceeded thetarget for intra-laboratory repeatability by more than the calculated multiplier, there would be a highlikelihood that this laboratory failed to adequately control variability during testing.
8. Test Method Data Quality
As indicated above, adherence to the 1998 draft test method was judged to be very good. This draft
adheres to and is in compliance with OECD GLP guidelines (see OECD 1998 publication: Principles ofGood Laboratory Practice and Compliance Monitoring, ENV/MC/CHEM(98)17).
9. Other Pertinent Scientific Reports and Reviews
At this time, it has not been possible to identify any other published or unpublished studies conducted byusing the proposed test method.

73ENV/JM/MONO(2005)14
10. Animal Welfare Considerations (Refinement, Reduction, and Replacement)
These considerations are not relevant to this method and ring-testing.

11. Practical Considerations (Evaluation of strengths and limitations of the test method)
Not relevant because an in vivo reference test method was not compared with the proposed new method.
See 4.0 above.
12. References
References are listed throughout this document.
13. Supporting Materials (Appendices)
None

74

ENV/JM/MONO(2005)14
Example 2

Report of the Final Ring Test of the Daphnia magna Reproduction Test.
OECD, Environment Directorate, OECD Environmental Health and Safety Publications Series on Testingand Assessment No. 6, 190 pp., by I. Sims, P. Van Dijk, J. Gamble, N. Grandy, and M. Huet (1997).
OECD, and Inter-Organization Programme for the Sound Management of Chemicals (IOMC)
1. Introduction and Rationale for the Proposed Test Method Validated
OECD Test Guidelines for Testing of Chemicals are periodically reviewed in relation to scientific progress
made since the TG first appeared. Scientific progress is difficult to succinctly define. However, it can
include development of new or modified procedures that are accepted by a consensus of ecotoxicologists
for incorporation into the test method, an accumulation of new test data and new ways of viewing andusing the method, limitations noted with the method as it is used more and more often, and thedevelopment of new technology applicable to the test method. For Test Guideline 202, Part II, Daphnia sp.
Reproduction Test (first adopted in April 1984), it was generally acknowledged that data from tests
performed according to this TG could be variable, and that there was a need to further update TG 202.
Results from a European Union (EU) ring-test conducted in 1985, which was based on a revision of the1984 OECD Test Guideline 202, Part II, also indicated an unacceptable level of variability in the interlaboratory results. Thirty-seven laboratories in 13 member countries participated. These results led to aseries of investigations (e.g., on daphnid genotype, food, and culture medium to use in the method) begun
in 1990, initially within the EU and later in OECD Member countries, to identify the sources of variability
and then eventually develop an improved Test Guideline. This work was equivalent to a Phase 1 orprevalidation activity.
A series of workshops were held in 1989, 1991, 1993, and 1995 to discuss daphnid toxicity and the
reproduction method, how to validate it, and assess the results of validation. OECD became involved after
the first workshop.
Results of the investigations were reviewed at the 1991 workshop, and it was determined that further work
would be necessary, i.e., a multi-staged ring-test would need to be completed. The first stage would be apilot or limited ring-test. In 1992 this study (also equivalent to Phase 1 or prevalidation) was conductedwith 36 laboratories, from 14 Member countries. Results of this work were reviewed at the 1993 workshop,
and planning for a second stage of Phase 2 full ring-test began.
In 1994 a full or final ring-test took place, based on a draft TG 202, Part II (dated February 1994) that wasdeveloped after the investigations above were concluded. Forty-eight laboratories in 16 Member countries(and the Czech Republic) participated in this ring-test. Details and results of the ring-test are given in the1997 Testing and Assessment Report cited above. Based on an analysis of the results of this activity at the
1995 workshop, the test method was again re-drafted. Additional discussion and review occurred in 19961997. The test method, as new OECD TG 211, was approved and adopted in September 1998.
Peer review of the pilot ring-test and full ring-test results occurred only among workshop members andattendees.

75ENV/JM/MONO(2005)14
A daphnid reproduction test is an important higher tier test, which can serve as an indicator or surrogate
test species for assessing the effects of pesticides and industrial chemicals on freshwater aquatic
invertebrates. This is an important test in hazard identification/assessment, classification and labelling, andin risk assessment.
2. Test Method Protocol Components
The principle of the test is to assess the effect of chemicals on the reproductive output of Daphnia magna.
Young female daphnids (the parent animals), aged less than 24 hours at the beginning of the test, areexposed to the test substance added to water at a range of concentrations. Test duration is 21 days and at
the conclusion of the test, the total number of living offspring produced per parent animal alive at the endof the test is assessed. Juveniles produced by adults that die during the test are excluded from thecalculations. Reproductive output of the parent animals can be expressed in other ways (e.g., number ofliving offspring produced per animal per day from the first day offspring were observed), but these should
be reported in addition to the total number of juveniles produced per parent alive at the end of the test. The
reproductive output of the animals exposed to the test substance is compared to that of the control(s) todetermine the lowest-observed-effect-concentration (LOEC) and the no-observed-effect-concentration(NOEC). In addition, and as far as possible, these data are analyzed by using a regression model toestimate the concentration that would cause an x% reduction in reproductive output, i.e., ECx (e.g., EC50,
EC20, EC10). Also, measured are survival of the parent animals and time to production of first brood.
Other substance-related effects on parameters such as growth (e.g., length), and possibly intrinsic rate ofincrease, may also be examined.
The method followed in the full ring-test was the draft OECD Test Guideline 202, part II, Daphna magna
reproduction test to be used in the final ring test, Appendix B in the 1997 OECD report cited above. Thismethod will not be discussed in detail here. Current complete details on this method, with subsequentchanges made since the Appendix B draft TG, are in the approved OECD TG 211 Daphnia magna
Reproduction Test, adopted on September 21, 1998.
3. Substances Used for Validation of the Proposed Test Method
Three test substances were used in the final ring-test, 3,4-dichloroaniline (DCA), cadmium chloride, andphenol. These chemicals were chosen because:
1) They have well-documented effects on Daphnia reproduction;2) They have different modes of toxic action;3) Stock solutions could be prepared without using solvents;
4) These chemicals would be relatively easy and inexpensive to analyze at the concentrations to be
used.
DCA was chosen to provide a link with the 1985 EU ring-test and the 1992 pilot ring-test. Cadmium waschosen to investigate the suitability of two culture media, which contain a known chelating agent, for
testing metals and metal compounds. Phenol was selected to assess the performance of the draft TG when
testing a “difficult” substance because it is readily biodegradable in the test system.
4. In Vivo Reference Data used for an Assessment of the Accuracy of the Proposed Test Method(where appropriate)
In vivo reference data and the method used to generate these data were not used to assess the accuracy of
the February 1994 method used in the full ring-test. Also, the February draft of TG No. 202, Part II,
differed substantially from the 1984 version, and it would thus have been difficult to compare one with the76

ENV/JM/MONO(2005)14
other. However, the results of this ring-test were compared with those from the 1985 ring-test, when theguideline was less defined, and were also used to investigate the intra or inter-laboratory variability.
5. Test Method Data and Results
The final ring-test thoroughly evaluated the performance of the February 1994 draft of TG 202, Part II.
Variability in the test results within and among laboratories was “low.” Significant improvements to the
TG have been made since 1984 as a result of this testing. In addition, it was possible to investigate various
ways of expressing reproductive output of the daphnids, and how to treat data from animals that die during
the test. Proposals for how these issues should be dealt with were made. Adherence to the instructions in
the February 1994 draft by the laboratories conducting the full ring-test was generally “very good.”“Most” laboratories were able to meet the recommended water quality criteria. Of the 98 experiments (52with DCA, 30 with cadmium, and 16 with phenol), the dissolved oxygen concentration, a critical test
condition, fell below the minimum in only one test. Also, 85% of the experiments were conducted within
the specified temperature range.
For DCA, approximately 50% of the NOEC and ECx values differed by a factor of 2 or less, over 75%
differed by a factor of 4 or less, and over 90% differed by a factor of 8 or less. For cadmium, 38% of the
NOECs differed by a factor of 2 or less and 62% differed by a factor of 8 or less. Cadmium EC50 s
differed by 27% and 45% respectively. For phenol, 45% of the NOECs differed by a factor of 3.2 or less,and 82% differed by a factor of 10 or less. Phenol EC50 s differed by 70% and 100%, respectively.
“Most” laboratories met the validity control performance criteria in the draft TG: 98% of the experiments
reported control mortalities of not greater than 20%, and 84% of experiments reported mean numbers oflive offspring per parent of at least 60.
6. Test Method Accuracy (Performance)
Comparisons with a reference test method were not made since in ecotoxicological testing there is no
generally accepted reference value. The closest to a reference value in ecotoxicology is where a weight-ofevidence approach is taken. Ecotoxicity test values derived from testing methods with “similar” species
may cluster together to give a mean toxicity value. A value from another method may fall within thiscluster. Thus, this approach may give some crude indication of method accuracy.
Accuracy has been defined by ICCVAM as determinations of concordance, sensitivity, specificity, positive
and negative predictivity, and false positive and negative rates. These various determinations were notmade in the Daphnia magna ring-testing.
7. Test Method Reliability (Repeatability/Reproducibility)
An important objective of the full ring-test was to obtain information on intra-laboratory variability, and toassess how this compares with inter-laboratory variability. Laboratories were asked to repeat the tests
under the same test conditions, where possible. Statistical analyses were performed to estimate intra-, andinter-laboratory variability for EC50 results using nominal concentrations of DCA only (the other two
chemicals had insufficient data sets for this analysis). The analysis was limited to EC50 values using total
juveniles as the response variable or test endpoint, for only laboratories that had performed repeat tests.
Results from ten laboratories were compared by using these criteria.
Intra or inter-laboratory variability (repeatability) was defined as the value below which the absolutedifference between two single test results obtained at the same laboratory, under repeatable conditions,77ENV/JM/MONO(2005)14
may be expected to lie with a probability of 95%. Intra-laboratory variance was estimated to be 12.36, andusing square roots to convert variances to SD gave a value of 3.5.
Inter-laboratory variability (reproducibility) was defined as the value below which the absolute difference
between two single test results obtained at different laboratories, under reproducible conditions, may be
expected to lie with a probability of 95%. Inter-laboratory variance was calculated to be 38.40, and usingsquare roots to convert variances to SD, gave a value of 6.2.
The repeatability and reproducibility values were calculated by multiplying the 3.5 or 6.2 values by thesquare root of 2 to give the standard error of the difference between two observations and then by 1.96 (the
95% two sided point of the standard normal distribution). The combined multiplier was calculated to be
2.8. Using this multiplier yielded a repeatability value of 10µg/L and a reproducibility value of 20 µg/L,with a ratio of 2. Thus, repeat results among laboratories are twice as variable as repeat results within alaboratory. Furthermore, two EC50 results from within the same laboratory cannot be considered to bedifferent unless they differ by more than 10 µg/L, and two results from different laboratories cannot be
considered different unless they differ by more than 20 µg/L. This ratio of 2 compares favourably withratios obtained from other ring tests. A fathead minnow ring-test had a ratio of 1.3, and a Daphnia 48 hour
acute toxicity ring-test had a ratio of 2.6.
8. Test Method Data Quality
As indicated above, adherence to the February 1994 draft method was generally “very good.” This draft
adheres to and is in compliance with OECD GLP guidelines (see OECD 1998 publication: Principles ofGood Laboratory Practice and Compliance Monitoring, ENV/MC/CHEM(98)17).
9. Other Pertinent Scientific Reports and Reviews
At this time, it has not been possible to identify any other published or unpublished studies conducted byusing the updated test method.
10. Animal Welfare Considerations (Refinement, Reduction, and Replacement)
These considerations are not relevant to this method and ring-testing.
11. Practical Considerations (Evaluation of strengths and limitations of the test method)
Not relevant because an in vivo reference test method was not compared with the updated new method.
See 4.0 above.
12. References
References are listed throughout this document.
13. Supporting Materials
None

78

ENV/JM/MONO(2005)14
Example 3
Validation, Special Study and “catch-up” Validation of In vitro Tests for Skin Corrosion Potential –The Human Skin Model Test
Summary: After a series of studies, in 1998, two in vitro tests for skin corrosivity, namely the rat skintranscutaneous electrical resistance (TER) assay and a test employing a human 3D reconstructed skinmodel, had been endorsed as scientifically valid by the European Centre for the Validation of Alternative
Methods (ECVAM, Joint Research Centre, European Commission). As a consequence of requirements
specified in EU Directive 86/609/EEC for the protection of laboratory animals, the two methods were in
the year 2000 adopted as one new Test Method B.40 ("In vitro Skin Corrosion") of Annex V to EU
Directive 67/548/EEC (on dangerous substances). However, at the more global OECD level the two
methods had not reached consensus, mainly because descriptions were too specific, requiring exactly theexperimental set-up's used in the validation studies. By the end of 2001, two OECD Extended NominatedExpert Consultations revealed that both methods could be described more general allowing for more
flexibility if additional conditions / requirements specified in the newly proposed Test Guidelines TG 430(TER) and TG 431 (human skin model) were met. Briefly, prevalidation-, validation, and additional special
studies were performed on a total of five in vitro tests for skin corrosion. In the prevalidation study threetests, the rat skin TER test, the CORROSITEX test, and the Skin² human skin model test were evaluated in
1994-1995. Fifty coded chemicals were tested in two to three laboratories. All tests showed sufficient
reproducibility and a promising concordance with in vivo skin corrosion potential. Experts reviewing theoutcome in ECVAM Workshop No 6 recommended proceeding to a formal validation study after method
refinements were made. The selection and acquisition of 60 new reference chemicals backed high quality
in vivo rabbit test data took almost a year. The formal validation study was then conducted with the threetests of the prevalidation study, plus a second skin model test (EPISKIN) in 1996-1997, and evaluated andpublished 1998. Whereas CORROSITEX showed a limited applicability and an insufficient predictivity,
and the Skin2 test protocol used (accepted by US DOT) was too insensitive, the TER test and the EPISKIN
test both met the predefined acceptance criteria. During the validation study, it was decided to perform an
additional small-scale special study: Since for the 60 test chemicals SAR data, in vivo data and in vitro
data (from the validation study) were available, it was decided to additionally determine pH and acidalkaline reserve, and apply the OECD tiered testing approach outlined in OECD TG 404. This study
showed that by SAR, pH and in vitro data 100% of the corrosive chemicals were detected. Since both Skin2
and EPISKIN became unavailable due to marketing decisions of the producers in 1996-1997, ECVAM
supported a new type of “catch-up” validation study. This catch-up study was designed to determine if
another human skin model (EpiDerm; available since 1992) could be used with a test protocol resembling
the one used for the successfully validated EPISKIN. The outcome of the study was almost identical withEPISKIN, confirming the robustness of the test design and the assumption that OECD Test Guideline 431
can be applied to different (including new) skin models.
1. Introduction and Rationale for the Proposed Test Method Validated
Skin corrosion refers to the production of irreversible tissue damage in the skin following the applicationof a test material as defined by the United Nations Globally Harmonised System for the Classification andLabelling of Chemical Substances and Mixtures (GHS). The assessment of skin corrosivity has typicallyinvolved the use of laboratory animals (rabbits). Concern for the pain and suffering involved with thisprocedure has been addressed in the 2002 revision of OECD Test Guideline 404 allowing for thedetermination of skin corrosion by using alternative in vitro methods.
A first step towards defining alternative tests that could be used for skin corrosivity testing for regulatory
purposes was the conduct of prevalidation studies (1). Following this, a formal, validation study of in vitro
methods for assessing skin corrosion (2)(3) was conducted (4)(5)(6). The outcome of these studies and79ENV/JM/MONO(2005)14
other published literature (7) led to the recommendation that two in vitro tests could be used for theassessment of in vivo skin corrosivity (8)(9)(10)(11): the human skin model test (this Method) and thetranscutaneous electrical resistance test (OECD Test Guideline 430). The validation studies have reported
that tests employing human skin models (5)(6)(7) are able to reliably discriminate between known skincorrosives and non-corrosives, in particular, if supported by physicochemical information (12).
2. Test Method Protocol Components
The mechanisms of skin corrosion in vivo are well understood. One mechanism is a destructive "digestion"of the skin barrier and underlying viable skin cells by pH extremes (acids and alkalines and their derivates),
the other main mechanism is the combination of high cytotoxicity / reactivity of a substance with high skinpermeability. In this case, the toxic substance quickly reaches the basal skin layers revealing a necrotic(irreversible) damage. Both in vivo mechanisms can be detected in a specific short term protocol of acytotoxicity test, measuring cellular viability in a human skin model (provided it has a well developed
stratum corneum barrier).
The test material is applied topically to a three-dimensional human skin model, comprising at least areconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by theirability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction
assay (13)) below defined threshold levels at specified exposure periods.
3. Substances Used for Validation of the Proposed Test Method
Prevalidation Study (1993-1994): Fifty test chemicals were selected on the basis of their availability andthe confidence with which they could be classified unambiguously as "corrosive"or "non-corrosive" (1).
Most of the test chemicals (25 corrosives, 25 non-corrosives) were commercially available.All test samples
were independently coded by Unilever ESL and codes were unknown to the participants. To ensure theintegrity of the raw data, test results were lodged with ECVAM prior to the code being broken.
Formal Validation Study (1996-1998): Sixty chemicals (30 corrosives / 30 non-corrosives) were selected
representing all chemical classes relevant to skin corrosion potential: organic acids, organic bases, neutral
organics, phenols, inorganic acids, inorganic bases, inorganic salts, electrophiles, and soaps/surfactants (2).
The main sources for the selection were in vivo data from ECETOC Report No. 66, but also in vivo data
sponsored from In Vitro International (IVI, Irvine, USA). All test samples were independently coded anddistributed by BIBRA Intl. and codes were unknown to the participants. To ensure the integrity of the raw
data, test results were lodged with ECVAM prior to the code being broken.
Special Study on OECD TG 404 Tiered Testing Approach (1997-1998): In the "special study" the same setof 60 chemicals as used in the formal validation study was evaluated for pH, and acid/alkaline reserve in ablinded manner (12).
Catch-up Validation Study (1997-1999): Fifty chemicals (25 corrosives / 25 non-corrosives) were used toadapt and refine the EpiDerm protocol and Prediction Model, the main selection criterion being the lowest
possible overlap of substances with the formal ECVAM validation study. In the final blind trial(performance assessment in three laboratories), a representative sub-set of the formal validation study wasused (7).

80ENV/JM/MONO(2005)14
4. In vivo Reference Data used for an Assessment of the Accuracy of the Proposed Test Method(where appropriate)
Prevalidation Study (1993-1994): In vivo data were obtained from in- house studies or from the scientific
literature, or by reference to the 1991 “Comité Européen des agents de Surface et leurs Intermediaries Organiques” (CESIO) classifications, or to manufacturers data. All animal tests were reported to have been
conducted in accordance with OECD Test Guideline 404. Corrosivity classifications were from threesources: animal data, hazard data sheets, or Annex 1 of the Dangerous Substances Directive (67/548/EEC)
The selection of the test chemicals was based primarily on the availability of the test material andassociated data. Thus, the criteria for selection were not as stringent as those employed by other groups (forexample, the European Centre for the Ecotoxicology and Toxicology of Chemicals [ECETOC ].
Formal Validation Study (1996-1998): An independent "Chemical Selection Sub-Committee" (CSSC) was
set up to guarantee that chemicals selected met the highest standards with regard to their in vivo
classification, supporting further evidence on corrosive/non-corrosive properties, chemical classes
represented, corrosivity subcategories represented, and avoidance of a majority of pH-extremes that could
be identified as corrosives by other means than a biological test system. In the CSSC the European
chemical industry (ECETOC) was represented, as well as a competent authority, an SAR expert, and thesponsor, ECVAM. Sixty chemicals (30 corrosives/30 non-corrosives) were selected representing all
relevant chemical classes: organic acids, organic bases, neutral organics, phenols, inorganic acids,
inorganic bases, inorganic salts, electrophiles, and soaps/surfactants. A chemical was only selected if at
least one original in vivo study report was available. The main source was data from ECETOC Report No.
66.
Reference Data proposed in OECD Test Guideline 431:
To allow future new "me too" skin models to be used in the context of the accepted OECD Test Guideline
431, twelve chemicals were selected by Experts in an OECD Extended Nominated Expert Consultation(11). Most of the chemicals listed are taken from the list of chemicals selected for the ECVAM
international validation study (3). Their selection is based on the following criteria:i) equal number of corrosive and non-corrosive substances;ii) commercially available substances covering most of the relevant chemical classes;iii) inclusion of severely corrosive as well as less corrosive substances in order to enablediscrimination based on corrosive potency;iv) choice of chemicals that can be handled in a laboratory without posing other serious hazards
than corrosivity.

81ENV/JM/MONO(2005)14
Reference Chemicals list:
1,2-Diaminopropane

CAS-No. 78-90-0

Severely Corrosive

Acrylic Acid

CAS-No. 79-10-7

Severely Corrosive

2-tert. Butylphenol

CAS-No. 88-18-6

Corrosive

Potassuim hydroxide (10%)

CAS-No. 1310-58-3

Corrosive

Sulfuric acid (10%)

CAS-No. 7664-93-9

Corrosive

Octanoic acid (caprylic acid)

CAS-No. 124-07-02

Corrosive

4-Amino-1,2,4-triazole

CAS-No. 584-13-4

Not corrosive

Eugenol

CAS-No. 97-53-0

Not corrosive

Phenethyl bromide

CAS-No. 103-63-9

Not corrosive

Tetrachloroethylene

CAS-No. 127-18-4

Not corrosive

Isostearic acid

CAS-No. 30399-84-9

Not corrosive

4-(Methylthio)-benzaldehyde

CAS-No. 3446-89-7

Not corrosive

5. Test Method Data and Results
For the data obtained in the prevalidation study (1) with the full thickness human skin model Skin² a
prediction model developed for classification into 3 corrosivity subcategories (I/II/III) according to UN
regulations for the transport of dangerous goods were applied, because this in vitro test had been acceptedin 1994 by the US DOT based on in house data submitted by the producer of the skin model. However, inthe prevalidation study the ability to correctly subcategorise corrosives could not be confirmed, and results
were therefore evaluated on the basis of ability to correctly separate the groups of skin corrosives from
non-corrosives (C/NC).
In the validation study (3), the two evaluated human skin model tests (Skin² and EPISKIN) used different
protocols and different prediction models. While the EPISKIN protocol and prediction model were closely
matching the design of the in vivo rabbit test (MTT reduction data after exposure of 3 min, 1 hr, and 4 hrs
were used to classify C/NC, or R34/R35/NC), for the Skin² test the US DOT accepted protocol andprediction model was used. Expectedly, due to the short exposure of only 10 seconds, the Skin² test was
100% specific, but showed an unacceptable low sensitivity (43%) and did therefore not meet thepredefined acceptance criteria for a validated skin corrosion test (3). Information given in the following
paragraphs on the outcome of this study is therefore only related to EPISKIN, which showed an acceptableperformance.
In the catch-up validation study, performed with the skin model EpiDerm (7), the original prediction model
to interpret data derived from a MTT reduction time course assay (14) “a chemical reducing the MTT
conversion after an exposure of less than 3 minutes is a corrosive” was turned into a prediction model
which was closer related to the validated EPISKIN test (“if the MTT conversion after 3 minutes exposure is
below 50% of untreated controls, the chemical is predicted corrosive”. In a second phase of this study (7),an additional 1 hr exposure was added, to obtain an increased test sensitivity, and a balanced positive andnegative prediction of > 80%. A third exposure time of 4 hrs (as used in EPISKIN) was not used, since it
did not contribute to an increase of test performance (7).

82ENV/JM/MONO(2005)14

6. Test Method Accuracy (Performance)
Compared to in vivo rabbit data, the following Cooper statistics (2x2 tables) performance measures wereobtained in the three studies (1)(3)(7):
Prevalidation study (Skin²):
Sensitivity:
64%, 96%, 84%(laboratory A, B, and C)
Specificity
76%, 88%, 76%(laboratory A, B, and C)
The performance in laboratory B was regarded acceptable, and in laboratories A and C promising toproceed to a formal validation study.
Validation Study (EPISKIN):
Sensitivity:
81% / 83%
(overall performance, prediction model A / B)
Specificity
80% / 80%(overall performance, prediction model A / B)
The performance of both prediction models was regarded acceptable (with a slight preference to prediction
model B) as the measures exceeded the predefined acceptance criteria of the best possible outcome
considering the variability of the in vivo reference data.
Catch-up Validation Study (EpiDerm):
Sensitivity:
72% / 88%
(overall performance, prediction model A / B)
Specificity
100% / 86%(overall performance, prediction model A / B)
The performance of prediction model A (using only data of 3 min exposure) was regarded not sufficient as
for a replacement test (however, valuable for positive screening). The performance of prediction model B(using data of 3 min and 1 hr exposure) was regarded excellent as the measures exceeded the predefined
acceptance criteria of the best possible outcome considering the variability of the in vivo reference data.

7. Test Method Reliability (Repeatability/Reproducibility)
To check repeatibility over time within one laboratory and reproducibilty between laboratories, in all threestudies (1)(3)(7) ANOVA was applied. In case of the formal validation study (3) the reproducibility waseven assessed for each of the 60 test chemicals. Repeatability within a laboratory over time was excellent
in all three studies and no significant differences were observed between independent tests. With regard to
reproducibility, the prevalidation study revealed a higher level of concordance (80%) between laboratories
B and C than between A and B, or A and C, indicating that the test protocol needs to be better defined andlaboratory trainings improved. In the two following studies, concordance between laboratories was almost
100% and ANOVA revealed that difference between laboratories had no impact on differentiating the test
chemicals by their corrosive potential.

8. Test Method Data Quality
Whereas the prevalidation study was performed applying GLP standards, the formal validation study andthe "catch -up-Validation Study were performed fully GLP compliant. However, none of the studies was
subjected to an internal or external study audits.

83ENV/JM/MONO(2005)14
9. Other Pertinent Scientific Reports and Reviews
Following the publication of the three studies (1)(3)(7), and the acceptance of the two skin model tests by
ECVAM (8)(9), ICCVAM evaluated the methods and published in 2002 a report on the EpiDerm,
EPISKIN and TER skin corrosion tests (15)
In 2004, ICCVAM published recommended Performance Standards to be met by new "me too" tests ofsimilar nature as CORROSITEX, TER, EpiDerm, and EPISKIN (16). The requirements to be met by new
test systems defined by ICCVAM are higher (with regard to the reference chemical number) than definedin OECD Test Guideline 431.
10. Animal Welfare Considerations (Refinement, Reduction, and Replacement)
For the rare purpose of only assessing skin corrosivity potential, the test may be regarded as Replacement
test. However, in almost all cases knowledge of skin irritation potential will be necessary as well. As longas no validated skin irritation test will be available, the test will function as part of the tiered strategy
shown in OECD TG 404, to screen out corrosive chemicals, so to act as Reduction and Refinement test.

11. Practical Considerations (Evaluation of strengths and limitations of the test method)
Chemical action by the test material on the vital dye may mimic that of cellular metabolism leading to afalse estimate of viability. This has been shown to happen when such a test material is not completely removed from the skin by rinsing (7). If the test material directly acts on the vital dye, additional controls
should be used to detect and correct for test substance interference with the viability measurement.
12. References(1)

Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., Esdaile, D.J., Gardner, J.R., Gordon,
V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Regnier,
J.F., Steiling, W., Walker, A.P., and Balls, M. (1995). A prevalidation study on in vitro skincorrosivity testing. The report and recommendations of ECVAM Workshop 6. ATLA 23, 219-255.

(2)

Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, A.P., and Worth, A.P. (1998). The
ECVAM international validation study on in vitro tests for skin corrosivity. 1. Selection anddistribution of the test chemicals. Toxic. in Vitro 12, 471-482.

(3)

Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J.,
Holzhutter, H.-G., and Liebsch, M. (1998). The ECVAM international validation study on in vitro
tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxic. in Vitro 12,483-524.

(4)

OECD (1996). Final Report of the OECD Workshop (Solna Report) on Harmonization of Validation
and Acceptance Criteria for Alternative Toxicological Test Methods, 62pp.

(5)

Balls, M., Blaauboer, B.J., Fentem. J.H., Bruner. L., Combes, R.D., Ekwall, B., Fielder. R.J.,
Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski. D., Spielmann,
H., and Zucco, F. (1995). Practical aspects of the validation of toxicity test procedures. The reportand recommendations of ECVAM workshops. ATLA 23, 129-147.

84

ENV/JM/MONO(2005)14(6)

ICCVAM (1997). Validation and Regulatory Acceptance of Toxicological Test Methods. A Reportof the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods, 105pp.

(7)

Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, P., Wilkins, S., McPherson, J.P.,
Wiemann, C., Kaufmann, T., Remmele, M. and Holzhütter, H.-G. (2000). The ECVAM
prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA 28, pp. 371-401.

(8)

ECVAM (1998). ECVAM News & Views. ATLA 26, 275-280.

(9)

ECVAM (2000). ECVAM News & Views. ATLA 28, 365-67(10) ICCVAM. (2001). Dermal corrosivity assays: EpiDermTM, EPISKINTM and Rat Skin Transcutaneous
Electrical Resistance. Background review document; public comments. Research Triangle Park,
NC: Interagency Coordinating Committee on the Validation of Alternative Methods/National
Toxicology Program Center for the Evaluation of Alternative Toxicological Methods.
http://iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf(11) OECD (2002) Extended Expert Consultation Meeting on The In Vitro Skin Corrosion Test
Guideline Proposal, Berlin, 1st –2nd November 2001, Secretariat's Final Summary Report, 27th
March 2002, OECD ENV/EHS, available upon request from the Secretariat(12) Worth AP, Fentem JH, Balls M, Botham PA, Curren RD, Earl LK, Esdaile DJ, Liebsch M (1998).
An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26: 709-720.
(13) Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63.
(14) Perkins, M.A., Osborne, R. & Johnson, G.R. (1996). Development of an in vitro method for skincorrosion testing. Fundamental and Applied Toxicology 31. 9 - 18(15) ICCVAM (2002) ICCVAM Evaluation of EPISKIN™, EpiDerm™ (EPI-200), and the Rat Skin
Transcutaneous Electrical Resistance (TER) Assay: In Vitro Test Methods for Assessing Dermal
Corrosivity Potential of Chemicals (NIH Publication No: 02-4502)
http://iccvam.niehs.nih.gov/methods/epiddocs/cwgfinal/cwgfinal.pdf(16) ICCVAM (2004) Recommended Performance Standards for In Vitro Test Methods for Skin
Corrosion (NIH Publication No: 04-510)
http://iccvam.niehs.nih.gov/methods/ps/ps044510.pdf

13. Supporting Materials
None

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Example 4
The Local Lymph Node Assay (LLNA)
1. BackgroundIn 1997, Drs. Frank Gerberick (Procter and Gamble, USA), David Basketter (Unilever, UK), and Ian
Kimber (Zeneca, UK) proposed to ICCVAM that the murine local lymph node assay (LLNA) should beconsidered for regulatory acceptance as a substitute for existing test methods used to assess dermal
hypersensitivity. The LLNA had undergone validation studies to characterize its utility and limitations(Basketter and Scholes, 1992; Basketter et al., 1991, 1994, 1996; Chamberlain and Basketter, 1996;
Gerberick et al., 1992; Kimber and Weisenberger, 1989; Kimber et al., 1989, 1990, 1991, 1995, 1998;
Loveless et al., 1996; Scholes et al., 1992). The sponsors asked ICCVAM to evaluate the validity of the
LLNA as a stand-alone substitute to guinea pig (GP) methods traditionally used to identify contact
allergens. The LLNA was already accepted as a screening test whereby a positive response would be
accepted, but a negative response would require GP testing. Their proposed use of the LLNA was thatboth negative and positive LLNA data should be accepted without the need for secondary testing. Thus,
chemicals that fail to elicit a positive response in the LLNA should be classified as lacking significant skinsensitizing potential and should be handled and labelled accordingly without the need for further
confirmation testing. The sponsors provide information supporting the validity of the LLNA as outlined in
the ICCVAM test method submission guidelines available at that time (ICCVAM, 1999).
2. Scientific Rationale and Mechanistic Basis of the LLNA
Allergic Contact Dermatitis (ACD) develops in two phases. In the first phase, sensitisation (induction
phase) occurs after initial skin contact with a sensitising agent. At the site of chemical contact, there is animmediate release of signalling factors and activation of the skin dendritic cells. The dendritic cells
process the chemical, and subsequently mature and migrate to the draining lymph node. It is this processthat induces the dendritic cells to become effective antigen presenting cells. Lymphocytes within thenodes, upon antigen presentation, undergo cellular proliferation. Proliferation is the endpoint evaluated in
the LLNA and the mechanistic basis of the assay. It also marks the first phase of ACD, the induction
phase (Selgrade et al., 2001).
Following proliferation, T lymphocytes are considered “primed” as they have a specific recall for thesensitizing agent. Upon additional exposure to the agent, an antigen specific response occurs which isreferred to as the elicitation phase. This second phase occurs only upon challenge with the specific agent.
There is an immediate release of inflammatory agents, in addition to immune specific mediators that cause
an inflammatory cell influx to the dermal site. Elicitation is a systemic response that can occur at locations
other than the original site of sensitization. The elicitation phase is characterised by erythema and oedema
and occurs 24 to 72 hours after the challenge exposure. This response is the endpoint assessed in
traditional GP tests (Selgrade et al., 2001).
3. Regulatory Rationale for the LLNA
ACD is associated with chemical exposure in the workplace and at home. An assessment of the potential
for chemicals to cause ACD is an important component of safety testing. Traditionally, GP tests have beenused for more than 65 years to assess the ACD potential of chemicals, pharmaceuticals, and consumer
products (Landsteiner and Jacobs, 1935; ICCVAM, 1999). While there are variations in these test methods,
the guinea-pig maximization test (GPMT) and the Beuhler Assay (BA) are used most commonly. Both ofthese tests rely on the sensitization and challenge phases of ACD, utilize 20 animals per test group, andrequire about one month to perform the assay. The GPMT has been criticized for the deleterious effect that86

ENV/JM/MONO(2005)14
the use of adjuvant has on treated animals; however, this test method is considered more “sensitive” thanthe BA. The endpoint measured for both GP methods is a visual assessment (subjective) of erythema at thechallenge location and requires substantial technical expertise (Marzulli and Maibach, 1996).
4. Description of the LLNA Test Method Protocol
The LLNA measures lymphocyte proliferation in the draining lymph nodes of mice topically exposed tothe test article using the incorporation of radioactive thymidine or iododeoxyuridine into DNA. The results
are expressed as a ratio (the stimulation index or SI) of the mean number of disintegrations per minute(DPM) for treated mice as compared to controls. Chemicals with a SI of 3.0 or more are considered
positive, and those with a SI less than 3.0 are considered negative. This scoring differs from the scoring in
GP assays in which a test substance is classified as positive based on the percentage of animals in a groupthat are responders (at least 15% in a non-adjuvant assay and at least 8% in an adjuvant test) (Marzulli and
Maibach, 1996). The measured proliferation in the LLNA is an essential biological response during theinduction phase of sensitization, and results from the increased proliferation of lymphocytes in the lymph
node due to the sensitizing chemical exposure. In contrast, as mentioned earlier, the currently accepted GP
assays, primarily the GPMT and the BA, rely on the elicitation phase of ACD by measuring skin reactivity
to a secondary challenge with the test substance. Furthermore, as mentioned earlier, endpointinterpretation in GP tests is subjective and requires substantial technical expertise.
The proposed detailed complete protocol necessary to perform the LLNA was provided. The basis for
protocol modifications made during development and validation was described, as was the rationale for
critical aspects of the protocol. Examples of other protocol elements described included: the basis for
decision criteria to classify a response as a positive or negative result; rationale for selection of the test
system (i.e. strain, sex, age, weight); rationale for the selection of positive controls; and basis for the dose
selection procedures.
LLNA and Reference Data: LLNA data were submitted for 209 chemicals representing a wide range ofchemical and product classes. The accuracy of the LLNA for predicting the outcome from standard ACD
tests was assessed by comparing LLNA results with reference data for 126 chemicals from standard GP
test methods. The accuracy of the LLNA for predicting human ACD was assessed by comparing LLNA
results with data from 39 chemicals tested in humans using the human maximization test, and 35 additional
chemicals included in human patch test allergen kits. The extent to which data were collected inaccordance with Good Laboratory Practice guidelines was described.
Test Method Performance: Calculations of performance were provided for sensitivity, specificity,
accuracy, positive and negative predictivity, and false positive and negative rates. The overall accuracy of
the LLNA compared to the guinea pig results was 86%. The accuracies of the LLNA and the guinea pig
tests, compared to human results, were both 72%.
Intra- and Inter-Laboratory Reproducibility: Data and analyses were provided to characterize the intraand inter-laboratory reproducibility of the LLNA. Three studies were provided, with each study involving
up to six different chemicals in three to six laboratories.
Animal Welfare Considerations: The extent that the LLNA reduced, refined, or replaced animal use
compared to the existing GP method was described. The basis for determining that the number of animals
used was the minimum number necessary to accomplish the testing objective was provided. The
submission stated that the LLNA required fewer animals than the traditional GP tests. It also stated that
the LLNA did not involve the pain and distress that might occur with procedures in the GP tests.

87ENV/JM/MONO(2005)14
5. ICCVAM Evaluation and Peer Review of THE LLNAICCVAM determined that the LLNA submission was sufficiently complete and that it was relevant to theneeds of agencies and agreed to proceed with an independent peer review panel evaluation of the LLNA.
An ICCVAM Immunotoxicity Working Group (IWG) composed of Federal government scientists wasformed to assist with the peer review evaluation and implementation process. Working grouprepresentatives had specific knowledge and experience applicable to the proposed test method, or werefamiliar with the current test methods used to generate data for ACD. NICEATM coordinatedcommunications, meeting preparation, document handling, literature reviews, statistical support, and final
report preparation. Public comment was encouraged throughout the process. A public docket wasestablished where all LLNA data and information considered by the Panel could be accessed. The public
Peer Review Meeting, availability of the test method submissions, and a request for public comment on the
LLNA were announced in a Federal Register Notice (63 FR 37405-37406).
6. LLNA Independent Peer Review Panel
The LLNA Panel consisted of 13 national and international experts in the field of immunotoxicology andallergic contact dermatitis (Dean et al., 2001). Panel members were from academia, industry, andgovernment, and had experience in biostatistics, toxicology, clinical medicine, and molecular immunology.
The international community was represented with Panel members from Denmark, Japan, and Norway.
All Panel members were required to sign a statement indicating that their involvement in the evaluation of
the LLNA did not constitute a financial or other conflict of interest. Panel members reviewed extensive
submission materials on the LLNA, participated in the public Peer Review Meeting, and prepared a written
report of their evaluation of the LLNA.
7. Panel Evaluation of the LLNA
The Panel was charged with evaluating the extent to which the ICCVAM validation and acceptance criteria
had been addressed. Questions were developed to ensure the completeness of the Panel’s evaluation withregard to test method description, test method performance, test method quality, determination of test
method reliability (repeatability/reproducibility), other scientific reviews, other considerations, and related
issues. Two overall questions were also proposed to the Panel: (1) Has the LLNA been evaluatedsufficiently and is its performance satisfactory to support its adoption as a stand-along alternative? and (2)Does the LLNA offer advantages with respect to animal welfare considerations (refinement, reduction, andreplacement)?
The Panel requested additional information and analyses that they considered necessary for their evaluation.
Examples of information requested by the Panel included original data, additional statistical analyses, andadditional information about the traditional GP assays. LLNA data and comments received from public
sources were provided to the Panel for their consideration.
8. Panel Meeting
The Panel convened on September 17, 1998, in Gaithersburg, Maryland, to review the LLNA in a public
meeting. Presentations were made on the ICCVAM criteria and process, the current regulatory testing
requirements for ACD, and the procedure for conducting the LLNA. Panel members then presented anddiscussed their evaluations, and presented their conclusions and recommendations. Public comment was
permitted at specific times during the meeting. The sponsors provided useful comments and responded toquestions from the Panel.

88ENV/JM/MONO(2005)14
9. Panel Report
The report of the LLNA Panel was published in 1999 (ICCVAM, 1999). Details on the LLNA Panel’s
conclusions and recommendations can be found also in Dean et al. (2001). The Panel unanimously
recommended the LLNA as a stand-alone alternative to GP tests, provided that some specificrecommendations on procedural aspects were incorporated (ICCVAM, 1999; Dean et al., 2001). In
addition, the Panel concluded unanimously that the LLNA provided animal welfare improvements
compared to currently accepted GP methods with respect to animal use, reduction, and refinement.
10. ICCVAM Test Method Recommendations
Following completion of the LLNA Peer Panel Report, the IWG developed and forwarded draft
recommendations on use of the LLNA to ICCVAM. The IWG concluded that the LLNA provided specificadvantages and potentially more accurate assessments of ACD compared to GP tests. One advantage isthat the interpretation of the LLNA is not affected by coloured agents, as may occur with GP tests.
Therefore, the LLNA was recommended as the preferred test method for coloured agents. Anotheradvantage is that the well-characterized biology of the mouse and availability of commercially produced
murine reagents will likely support future developments and expanded applications of this assay. The
IWG noted that there may be regulatory situations that require chemicals to be tested in a clinically- orcommercially relevant solvent or product formulations. It was noted that some testing situations will
require continued use of the traditional GP test methods. These include the evaluation of metals or metalcompounds, strong irritants, and laboratory situations where use of the LLNA is not feasible due to its
current requirement for the use of radioisotopes. The IWG acknowledged that, with future development
efforts, the LLNA holds great potential for expanded applications and use. ICCVAM endorsed the IWG
and Panel recommendations and forwarded them to the Director, NIEHS, who in turn sent them to theparticipating ICCVAM agencies for regulatory acceptance consideration.
11. Acceptance of the LLNA by Federal Agencies
The U.S. Environmental Protection Agency (EPA), Food and Drug Administration (FDA), andOccupational Safety and Health Administration (OSHA) announced their acceptance of the LLNA as astand-alone test method at the October 1999 meeting of the NTP Advisory Committee on Alternative
Toxicological Methods (ACATM). They agreed that the LLNA can be used to assess the ACD potential ofchemicals and products when used in accordance with the recommendations in the LLNA Panel Report.
The acceptance process for each agency varied often due to the specific nature of the products and agentsregulated by each agency.
U. S. EPA Consideration of the LLNA
The U.S. EPA accepted the ICCVAM position on the validity of the assay and supports its use as the first
option for the identification of chemicals with contact sensitizing potential. U. S. EPA guidelines for theevaluation of chemicals for contact hypersensitivity potential are currently being revised to incorporate the
LLNA as an acceptable method that can be used to meet U. S. EPA requirements. The U.S. EPA plans to
identify situations in which the LLNA should be the recommended primary test, and situations in which
the assay should not be recommended because of limitations relating to particular agents and regulatory
situations.
FDA Consideration of the LLNA
The FDA reviewed the LLNA on a Center-by-Center basis. The Center for Drug Evaluation and Research(CDER) concluded that the LLNA is an acceptable stand-alone alternative to the GPMT for hazard
89ENV/JM/MONO(2005)14
identification of dermally applied contact sensitizing agents within the limitations acknowledged by the
Panel. The Center for Biologics Evaluation and Research (CBER) concluded that the LLNA is anacceptable alternative to the GPMT. CBER proposes to recommend the LLNA when skin sensitization
data are necessary and appropriate. The Center for Veterinary Medicine (CVM) concluded that the LLNA
is an acceptable alternative, but has no need to include the method in its testing guidelines. On the rareoccasions when a veterinary drug is tested for hypersensitivity potential, preference is given to the use of
the most appropriate test species. The Center for Devices and Radiological Health (CDRH) concluded that
the LLNA is acceptable with qualifications similar to those noted by the Panel. Additionally, CDRH
indicated that proper positive and negative controls for testing extracts of materials/devices should be
included in the testing paradigm. The Center for Food Safety and Applied Nutrition (CFSAN) concluded
that the LLNA is a valid, stand-alone alternative to the GPMT for detecting ACD mediated by food andcolor additives and cosmetics, within the limitations expressed by the IWG.
OECD Adoption of the LLNA
The LLNA was adopted as Test Guideline 429 in April 2002 by the Test Guidelines Programme of theOrganisation for Economic Co-operation and Development (OECD). The draft guideline was distributed
for review and commentary by the 30 member countries of the OECD in November 2000. The ICCVAM
Panel Report on the LLNA was distributed with the proposed guideline to substantiate its scientific validity.
12. Implementation
ICCVAM, in conjunction with the International Life Sciences Institute (ILSI) Health and Environmental
Sciences Institute’s (HESI) Alternatives to Animal Testing Technical Committee, organized a one-and-ahalf day training workshop on the LLNA in January 2001. The purpose of the workshop was to familiarize
the regulated community with the LLNA, and the manner in which agencies expect the method to be
conducted and interpreted. The workshop was designed for scientists from industry, government, andacademia who had an interest in learning more about the LLNA. The primary objective of the workshop
was to assist these participants in gaining a practical understanding of the theory and application of the
LLNA.
The workshop provided information on the scientific basis of the assay and its development and validation.
Practical conduct of the assay was emphasized including instruction on standard protocols and datainterpretation. A case study exercise provided workshop participants an opportunity to evaluate data andto apply knowledge gained from the lecture. This exercise allowed for discussion of specific scientific andregulatory issues and identification of assay advantages and limitations. Constructive dialogue between theindustry and regulatory agency staff at this workshop should expedite the implementation and global
acceptance of the LLNA.
The rigorous and comprehensive review provided by the ICCVAM process afforded government
regulatory agencies reasonable assurance of the usefulness and limitations of this new method.
13. REFERENCES
Basketter, D.A., and Scholes, E.W. (1992). Comparison of the local lymph node assay with the guinea pig
maximization test for the detection of a range of contact allergens. Food Chem. Toxicol. 30, 65-69.
Basketter, D.A., Scholes, E.W., and Kimber, I. (1994). The performance of the local lymph node assay
with chemicals identified as contact allergens in the human maximization test. Food Chem. Toxicol. 32,
543-547.

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Basketter, D.A., Gerberick, G.F., Kimber, I., and Loveless, S.E. (1996). The local lymph node assay—A
viable alternative to currently accepted skin sensitisation tests. Food Chem. Toxicology 34, 985-997.
Basketter, D.A., Scholes, E.W., Kimber, I., Botham, P.A., Hilton, J., Miller, K., Robbins, M.C., Harrison,
P.T.C., and Waite, S.J. (1991). Inter-laboratory evaluation of the local lymph node assay with 25
chemicals and comparison with guinea pig test data. Toxicol. Methods 1, 30-43.
Chamberlain, M., and Basketter, D.A. (1996). The local lymph node assay: status of validation. Food
Chem. Toxicol. 34, 999-1002.
Gerberick, G.F., House, R.V., Fletcher, E.R., and Ryan, C.A. (1992). Examination of the local lymph
node assay for use in contact sensitization risk assessment. Fundam. Appl. Toxicol. 19, 438-445.
ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods). (1999). The
Murine Local Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential ofchemicals/compounds. NIH Publication No. 99-4494, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina.
ICCVAM. (Interagency Coordinating Committee on the Validation of Alternative Methods). (1997).
Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency
Coordinating Committee on the Validation of Alternative Methods. NIH Publication 97-3981. National
Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. Available on theInternet at http://ntp-server.niehs.nih.gov/htdocs/ICCVAM/iccvam.html.
Kimber, I., and Weisenberger, C. (1989). A murine local lymph node assay for the identification ofcontact allergens. Assay development and results of an initial validation. Arch. Toxicol. 63, 274-282.
Kimber I., Hilton, J., and Botham, P.A. (1990). Identification of contact allergens using the murine local
lymph node assay: Comparisons with the Buehler occluded patch test in guinea pigs. J. Appl. Toxicol. 10,
173-80.
Kimber, I., Hilton, J., Botham, P.A., Basketter, D.A., Scholes, E.W., Miller, K., Robbins, M.C., Harrison,
P.T.C., Gray, T.J.B., and Waite, S.J. (1991). The murine local lymph node assay: Results of an interlaboratory trial. Toxicol. Lett. 55, 203-213.
Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L., House, R.V.,
Ladics, G.S., Loveless, S.E., and Hastings, K.L. (1998). Assessment of the skin sensitization potential of
topical medicaments using the local lymph node assay: An inter-laboratory exercise. J. Toxicol. Environ.
Health 53, 563-579.
Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., Ladics,
G.S., Loveless, S.E., House, R.V., and Guy, A. (1995). An international evaluation of the murine local
lymph node assay and comparison of modified procedures. Toxicology 103, 63-73.
Kimber, I., Hilton, J., and Weisenberger, C. (1989). The murine local lymph node assay for identificationof contact allergens: A preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact
Derm. 21, 215-220.
Landsteiner, K., and Jacobs, J. (1935).
compounds. J. Exp. Med. 61, 643-656.

Studies on sensitization of animals with simple chemical91

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Loveless, S.E., Ladics, G.S., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., House, R.V.,
Hilton, J., Dearman, R.J., and Kimber, I. (1996). Further evaluation of the local lymph node assay in the
final phase of an international collaborative trial. Toxicology 108, 141-152.
Marzulli, F.N., and Maibach, H.I. (Eds.).
Washington, D.C.

(1996).

Dermatotoxicology, 5th Ed.

Taylor & Francis,

Sailstad, D., Hattan, D., Hill, R., and Stokes, W. (2000). The first method evaluation by the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM): The murine local lymph
node assay (LLNA). In Progress in the Reduction, Refinement and Replacement of Animal
Experimentation (M. Balls, A.-M. van Zeller, and M.E. Halder, Eds.), pp. 425-433. Elsevier Science,
Amsterdam.
Scholes, E.W., Basketter, D.A., Sarll, A.E., Kimber, I., Evans, C.D., Miller, K., Robbins, M.C., Harrison,
P.T.C., and Waite, S.J. (1992). The local lymph node assay: Results of a final inter-laboratory validation
under field conditions. J. Toxicol. Appl. Pharmacol. 12, 217-222.
Selgrade, M.J.K., Germolec, D.R., Luebke, R.W., Smialowicz, R.J., Ward, M.D., and Sailstad, D.M.
(2001). Immunotoxicity. In Introduction to Biochemical Toxicology, 3rd Edition (E. Hodgson and R.C.
Smart, Eds.), pp. 597-561. J. Wiley and Sons, NY, NY.
Stokes, W., and Hill, R. (2000). The role of the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) in the evaluation of new toxicological testing methods. In Progress in the
Reduction, Refinement and Replacement of Animal Experimentation (M. Balls, A.-M. van Zeller, and M.E.
Halder, Eds.), pp. 385-394. Elsevier Science, Amsterdam.
Sailstad, D., Hattan, D., Hill, R., and Stokes, W.S., Evaluation of the Murine Local Lymph Node Assay(LLNA) I: The ICCVAM Review Process. Regul. Toxicol. Pharmacol. 34(3):249-257, 2001.
Dean, J., Twerdok, L., Tice, R., Sailstad, D., Hattan, D. and Stokes, W.S., Evaluation of the Murine Local
Lymph Node Assay (LLNA) II: Conclusions and Recommendations of an Independent Scientific Peer
Review Panel. Regul. Toxicol. Pharmacol. 34(3):258-273, 2001.
Haneke, K., Tice, R., Carson, B., Margolin, B., and Stokes, W.S., Evaluation of the Murine Local Lymph
Node Assay (LLNA): III. Data Analyses Completed by the National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). Regul. Toxicol.
Pharmacol. 34(3):274-286, 2001.
USC (United States Code) NIH/National Institutes of Health Revitalization Act. Public Law 103-43. 42
USC Section 205. Washington, DC: US Government Printing Office, 1993.
ICCVAM Authorization Act of 2000, Public Law 106-545, 106th Cong., 2nd sess. (December 19, 2000).

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Example 5
The Up-and-Down Procedure for Acute Oral Toxicity (TG 425)
1. Introduction
A US team of toxicologists and statisticians developed an improved version of the up-and-down procedure
as one of the replacements for the traditional acute oral toxicity test formerly used by OECD Member
countries to characterize industrial chemicals, pesticides and their mixtures. This method, OECD Test
Guideline 425 (2001), for applications that use the LD50, achieves significant reductions in animal use. It
employs sequential dosing, together with use of computer-assisted computational methods during theexecution and calculation phases of the test. Staircase design, a form of sequential test design, can beapplied to acute toxicity testing with its binary experimental endpoints (yes/no outcomes). The staircase
design used in the Test Guideline builds in efficiencies by concentrating most doses near the region of theLD50, and by using flexible or adaptive stopping rules to accommodate a variety of chemicals. The upand-down procedure provides a point estimate of the LD50 and approximate confidence intervals, inaddition to observed toxic signs, for the substance tested. However, when assumptions about standard
deviation of the test population diverge significantly from actual values, point estimates of the LD50 may
not be possible. In these cases, an LD50range estimate is provided. The test method does not provide
information about the dose-response curve.
2. History
In light of the heavy use of animals in traditional test methods, R.D. Bruce (1985) applied the up-and-down
procedure, a sequential test design, to acute toxicity testing. The test design assumed a value for sigma, thestandard deviation of the curve, which is suitable for certain industrial chemicals.
The Up and Down technique was evaluated in laboratory studies in 1987. Subsequently, two other studies
were done comparing the results of the up-and-down procedure to those using traditional LD50 test
methods, for 35 substances (Lipnick et al.,1995)
The goals of the United States design team were to fulfil the OECD mandate to develop a robust test thatwould be applicable to a wide variety of pesticides, industrial chemicals, and their products. The test
should have the ability to be performed, and for results to be calculated, without the need to assume a value
for the standard deviation (SD). Computer simulations were used to evaluate the performance of theprevious version of the OECD Test Guideline (OECD TG 425, 1998) and to determine appropriatechanges to optimize the method's performance without actually testing animals in the laboratory.
3. Rationale for Experimental Design Used in Test Guideline 425
Sequential test designs, in contrast to test designs with fixed sample size involving replicate sampling, canachieve efficiencies in the number of test samples needed by sampling one or a few test subjects at a time
until just enough measurements are made to evaluate the experimental endpoint of concern with the desired
precision. In general, sequential designs can be applied to either qualitative (yes/no) or quantitative
outcomes, but only the former are considered in this paper. Staircase designs permit trials to rapidly
converge on the region of interest, such as the ED50 or LD50 in a toxicity test.
For suitable applications in toxicology, the use of sequential design and non-traditional calculation
methods can lead to reduction of animal usage, while maintaining the ability of the test to measure desired
experimental results. Acute toxicity testing, which measures the adverse effects that occur within a short93ENV/JM/MONO(2005)14
time of administration of single dose of a chemical, is one such candidate for the application of sequential
design.
4. Test Description
The up-and-down procedure for acute oral toxicity involves testing young adult animals one at a time in astaircase fashion. Unless the conditions for terminating the test have been reached, the next dose level
depends on the results in the previous animal. If that animal survived, then the next dose is higher, while if
the last animal died, then the next dose is lower. This permits the experiments to converge on the region of
the LD50, which is the inflection point corresponding to the median response of the log-normal doseresponse curve.
The OECD TG 425 (2001) uses sequential dosing and calls for testing to be performed in a single sex toreduce variability in the test population. Animals are tested individually in a staircase fashion using a dose
progression and starting dose based on characteristics of the chemical being evaluated. A flexible oradaptive stopping rule limits the number of animals in the main test, while allowing the method to be
applied to chemicals with a wide range of slopes of the dose-response curve. A computer program isavailable to determine if conditions for terminating the test have been reached. Guidance is provided foruse of all available information to determine initial dosing and dose progression spacing. Initial doses
should be set at sub-lethal levels and dose progression based on approximate slope. However, the TG
includes recommendations for default values for starting dose and dose progression for use in the absenceof initial information. As in the traditional acute test, this replacement up-and-down procedure provides
for clinical observations over 14 days. While the statistical procedures are more complicated than those fora non-sequential test, much of the complexity can be handled by computer software.
In addition, a sequential limit test using up to five animals has been substituted for the ten-animal batch
limit test. Sequential observations permit a separate decision about the contribution of each animal to thedetermination of whether the LD50 lies above or below the limit dose.
5. Use of Statistical Simulation for Development and Validation of the Up-and-Down Procedure
By varying assumed values of the LD50 and the SD for a hypothetical set of animals exposed to a toxic
chemical, it is possible to have a computer generate responses from a randomly chosen sample of the
hypothetical population. A computer can use these hypothetical responses to simulate the results fromthousands of small samples of the underlying population. Since the underlying mean and SD of the test
population is known, these simulations then can be used to determine if changes in the test design would
improve the ability of the up-and-down procedure test to estimate the mean and SD. By varying the SD
assigned to the hypothetical set of animals, it is possible to simulate the degree of variation in theexperimental sample’s response that would occur because of animal-to-animal, inter-, and intra-laboratory
species, strain, sex, age, and housing variability. Such simulations have shown that the new samplingtechnique used in the up-and-down procedure has a much better chance of placing the estimated LD50
close to the value that was used in defining the underlying hypothetical population even when the starting
dose is inappropriate. This type of comparison would not be practical using actual animal tests, since itwould be impossible to determine whether each small sample tested is providing correct or incorrect
estimates of the underlying population.
Actual animal testing was not necessary for determination of the validity of the new statistical design. It is
not appropriate or possible to compare changes in sampling technique or to assess the ability of a newstatistical design to accurately estimate the mean and SD of the population based on the results of a few
runs of a test. This is because there is no way to determine the goodness of fit of the statistical procedurefrom a few samples. However, computer simulations can be used to compare the results of thousands of94

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individual hypothetical tests. By using a large series of such simulations varying starting dose, dose
progression and assumptions about mean number and SD of the underlying population, it is possible to test
how often a new statistical sampling technique will accurately estimate the LD50 and SD of the population.
Simulations showed that the number of test subjects needed to provide an acceptable degree of accuracy
depends, in part, on the slope of the dose-response curve of the test population. However, in most cases,
the slope is not known in advance of testing. Therefore, in order to allow the up-and-down method to be
applied to a wide variety of chemicals with reasonable reliability, a flexible stopping rule, using criteria
based on an index related to the statistical error, was developed and incorporated into the test. For
chemicals with higher slopes, the stopping rules will be satisfied with four animals after the first reversal.
Additional animals may be needed for chemicals with dose-response slopes below 4. In the interest ofanimal welfare, testing in any case is terminated at 15 animals. Simulations have shown that the averageanimal use is expected to be seven to nine animals per test.
6. Performance of the Revised TG 425
The revised OECD Test Guideline 425 (OECD 2001) has improved performance for prediction of the point
estimate of lethality (LD50) and confidence intervals for chemicals with wide variability of response
characteristics, even when the approximate LD50 and dose-response slope are not known.
Theefficiencies of the new up- and-down dosing routine allow the LD50 to be estimated using relatively few
animals. However, the Test Guideline does not provide for determination of the slope of the dose-response
curve.
Although flexible stopping rules allow the up-and-down procedure to be applied to test materials with awide range of slopes, for optimum performance of the up-and-down procedure, the dose progression usedshould be based on an accurate estimate of the SD. In addition, to account conservatively for any bias in
the LD50 estimate, it is essential that dosing be initiated below the actual LD50. Setting initial doses atsublethal levels also ensures that LD50 values are not underestimated while reducing distress in the
animals.
In traditional LD50 tests, the stated confidence interval, for example 95%, is exactly what is calculated.
However, for the up-and-down procedure, the algorithm used to compute 90%, 95%, or 99% profile
likelihood confidence intervals is not exact but approximate, so that in some situations, the statedconfidence interval will not provide the desired coverage or may provide more than the desired coverage.
Simulations indicate that actual coverage for the nominal 95% confidence interval falls below 95% when
the slope is shallow and above 95% when slopes are very steep. The nominal 95% confidence interval will
have coverage of at least 90% if the slope is 2-4 or greater (SD 0.25-0.5 or smaller). For most situations,the coverage will be better than 90% if the slope is 2 or greater. Coverage will be 80% or better if the slope
is at least 1. For slopes as low as 0.5, the coverage may be as low as 70%.
The up-and-down procedure does not allow for characterization of the slope since efficient management ofdoses toward the region of the LD50 does not provide enough doses in the wings of the dose-response
curve. If slope is needed, another method would be used. The revised up-and-down procedure Test
Guideline can be used for chemicals with a wide variety of actual dose-response slopes and can be used forhazard classification and certain other hazard and risk assessment purposes. The five-animal sequential
plan used in the limit test produces results that are almost as good as those for the present ten-animal fixedsample plan, while averaging three to five animals per test. That represents a substantial reduction ofanimal subjects over the ten-animal fixed sample plan.

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7. Peer Review
The revised Test Guideline was peer reviewed by independent panels of experts convened by theInteragency Coordinating Committee on the Validation of Alternative Methods (ICCVAM 2001) and by
the EPA Scientific Advisory Panel (EPA 2001).

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